DON from field to glass

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

Topics 1. DON a Fusarium Mycotoxin 2. Fusarium Head Blight of Barley and Wheat 3. Reducing the DON Risk from Field to Malthouse 4. Fusarium and Mycotoxins in Malting 5. Fusarium and Mycotoxins in Brewing 6. Testing of Mycotoxins 7. Conclusions

DON is a mycotoxin Mycotoxins are: Low molecular weight natural products Produced by filamentous microfungi Predominantly secondary metabolites Toxic to vertebrates in low concentrations

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)

Mycotoxins produced by Fusarium species Gibberella ear rot of corn Fusarium graminearum G.C. Bergstrom

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

FHB Produces Several Trichothecenes CH 3 O H R 1 O R 5 CH 3 R 4 R 3 CH 3 R 2 H 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

Proportion of trichothecene genotypes of Fusarium graminearum in 2011 and 2012 n = 152 n = 90 n = 87 n = 101 n = 128 n = 93 n = 95 3-ADON 15-ADON Gary C. Bergstrom, Cornell University Bergstrom et al. unpublished

Mycotoxin chemotypes of Fusarium graminearum in wheat in 2007 2011 Survey in New York 15% 3-ADON: 85% 15-ADON Same as in 2007 Schmale, Wood Jones, Cowger, Bergstrom, and Arellano. 2011. Plant Pathology 60:909 917

Mycotoxin production on wheat and maize is widely variable among isolates 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. Wheat kernels DON, 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. Maize kernels Kuhnem, P.R., Del Ponte,E.M., Dong, Y,. and Bergstrom, G.C. Phytopathology 22 Oct 2014 First Look Gary C. Bergstrom, Cornell University

Mycotoxin genotype prevalence in three NYS locations Aurora 2012 Aurora 2013 Willsboro 2013 N=281 N=428 N=319 Belmont 2013 GENOTYPE LAND USE N=379 15 ADON 3 ADON G.C. Bergstrom

Trichothecene genotype frequency by niche in New York in 2013 Kuhnem, Del Ponte, and Bergstrom See Poster No. 29

Fusarium head blight - the problem 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 grain Food and beverage products Baby food products Current US FDA Guidelines Current EC Guidelines Proposed FAO- Codex- Alimentarius 2.0 1.0 - - 0.5 0.1-0.5 0.1

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 Since 1999: $61.9 Million invested in research and outreach 1,628 research projects www.scabusa Involvement of 174 scientists

2015 Spring Malting Barley Integrated Fusarium Management Trial variety Powdery Mildew % Leaf Blotch % FHB Incidence % FHB Severity % FHB Index % DON ppm 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 Spray with an effective triazole based on risk of FHB. Effective spray window from full head emergence through the next 5-7 days Early June Late May Early May April

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% prothioconazole 19% & tebuconazole 19% 48 43 52 42 tebuconazole 38.7% 40 23 propiconazole 41.8% 32 12 Michael Wunsch *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 Powdery Mildew % Leaf Blotch % FHB Incidence % FHB Severity % FHB index % DON ppm 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% (EC 50 ) EC 50 = 8.09 mg/l Gary C. Bergstrom, Cornell University

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 Gary C. Bergstrom, Cornell University

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????? Resistant Cultivars Fungicides / Prediction Tools Cultural Practices 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 Ascospores Macroconidia avoiding or limiting exposure of cereal spikes to spores.

Management of overwintered cereal residues: Regional impact and benefits in individual wheat fields FHB severity declined during era of the moldboard plow, 1940s through 1970s Less debris decomposition and higher inoculum pressure in cold winter regions Regional increases in FHB, predominance of Fusarium graminearum as causal fungus associated with increased acreage of corn

Horizontal transport of spores over km distances in 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 100 ft Clonal source 3% Control % Clones Suggested a 3 to 6 meter (10 to 20 feet) interplot to avoid significant interplot interference from inoculum-bearing cereal residue Keller, Waxman, Bergstrom, and Schmale. 2010. Plant Disease 94: 1151 1155.

Commercial-scale wheat after corn strip trials (no-till vs moldboard-plowed) in seven states (2011-2013) Collaborators: Carl Bradley, Ann Hazelrigg, Don Hershman, Martin Nagelkirk, Laura Sweets, Stephen Wegulo Gary C. Bergstrom, Cornell University

Commercial-scale wheat after corn strip trials (no-till vs moldboard-plowed) experimental design Aurora, New York Mead, Nebraska

Average increase in DON of 22% (0.24 ppm) associated with no-till corn residue in wheat strips DON (ppm) 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 Moldboard Plow No till Chisel Plow 2013 * 2011 2012 * * * * IL KY MO NE NY VT MI IL KY MO NE NY VT MI IL NY NE Average 17% (0.38 ppm) increase when background level > 0.50 ppm

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? No single answer for all environments and cropping systems. Resistant Cultivars? Fungicides / Prediction Tools Cultural Practices For wheat/barley within corn-growing regions in the north central and northeastern U.S., generally less than 30% contribution to DON reduction.

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. Ranking of Grain Quality Issues by Growers #1 Ranking contribution #2 Ranking contribution #3 Ranking contribution #4 Ranking contribution 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 Dark molds are saprophytes not associated with symptoms Darkened florets indicative of FHB Pink Fusarium colonies grow from symptomatic florets

Survey of 40 commercial barley fields in 2015 Soil Tests Malt Quality DON & Fusarium Content Foliar Disease Acknowledgements: Kevin Ganoe, Mike Stanyard, Justin O Dea, Aaron Gabriel, Jaime Cummings

5.0 DON 4.5 4.0 3.5 3.0 ppm 2.5 2.0 1.5 1.0 0.5 0.0 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 Sample Number

DON 25 20 16 20 2014 2015 Samples 15 10 9 11 5 6 5 0 Less than 1 1 2 More than 2 ppm

Different DON results by ELISA vs chromatography 7 DON Comparison on 40 field samples measured by ELISA and GC MS DON (ppm) 6 5 4 3 ELISA GC-MS 2 1 0 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 Sample # G.C. Bergstrom

How good a predictor is grain DON of viable Fusarium content? DON vs Fusarium kernels 7 70 6 60 DON (ppm) 5 4 3 2 50 40 30 20 % Fusarium kernels 1 10 0 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 Sample # 0 UMN DON % Fusarium kernels G.C. Bergstrom

Viable Fusarium content before, during, and after craft malting? Grain Malt G.C. Bergstrom

Niagara Malt Pioneer Malting Inc. Queen City Malting NY Craft Malt Empire Malt East Coast Malts Empire Malt Farmhouse Malt Subversive Malting

On to the malt house Madison Co. NY 6/24/14 G.C. Bergstrom

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

Grain Cleaning Solutions to FHB and DON: 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. Maltster

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-control agent 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/gui dancedocumentsregulatoryinformation/chemicalc ontaminantsmetalsnaturaltoxinspesticides/ucm120 184.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 Beer Assumptions: 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 Testing Testing 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 Testing Testing 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/pa ges/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_donanalysis.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_s ampling.pdf