DON from field to glass Gary Bergstrom and Paul Schwarz Cornell University and North Dakota State University Great Lakes Hops and BarleyConference Traverse City, MI March 16‐17, 2016
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
Gary C. Bergstrom, Cornell University
What should occur as a consequence of these findings?
Gary C. Bergstrom, Cornell University
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)
Gary C. Bergstrom, Cornell University
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
Fusarium and Mycotoxins in Malting
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
Fusarium and Mycotoxins in Malting
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 and Mycotoxins in Malting
Fusarium on barley: symptoms of FHB are not always visible
Fusarium and Mycotoxins in Malting
Fusarium on germinating barley
Fusarium and Mycotoxins in Malting
• 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.
Fusarium and Mycotoxins in Malting
• However, this only works to a point.
• DON levels in more heavily infected samples generally will not be reduced to satisfactory levels by steeping.
Fusarium and Mycotoxins in Malting
• 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
Fusarium and Mycotoxins in Malting
• 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.
Fusarium and Mycotoxins in Brewing
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)
Fusarium and Mycotoxins in Brewing
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
Solutions to FHB and DON:Maltster
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.
Solutions to FHB and DON:Maltster
• 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?
Solutions to FHB and DON:Maltster
• 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
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
DON: Beer and Food Safety
• 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.
DON: Beer and Food Safety
• 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/
DON: Beer and Food Safety
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
DON: Beer and Food Safety
• 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