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Ontario Grain Farmer Magazine

Mitigating DON impacts on the Ontario corn industry

Principal Investigator: Art Schaafsma, Dave Hooker, Albert Tenuta

Research Institution: University of Guelph

Timeline: November 2018 – January 2023

Objectives:

  • Evaluate corn hybrid resistance to Gibberella ear rot (GER) and DON accumulation and identify mycotoxin dynamics across hybrids and environments.
  • Develop in-season best management practices (BMPs) for DON mitigation.
  • Develop sampling and testing BMPs for DON.
  • Identify BMPs for effective storage management of high-DON corn – a case study with grain bags.
  • Coordinate a grinder pilot project to effectively carry out the recommended sampling protocol for DON-contaminated grain.
  • Host an Ontario Grains Researcher Forum to identify research gaps and collaboration opportunities.
  • Knowledge transfer of project findings and recommendations.

Impacts:

  • Better understanding of corn hybrid and inbred resistance to DON accumulation will improve the quality of actionable information for farmers and the value chain.
  • Development of a better resistance screening protocol will help with the development of new hybrids with improved resistance to DON accumulation, as well as reduce the proportion of highly susceptible commercial hybrids in the marketplace. This will limit losses and quality downgrades to farmers and the industry and will provide assurances to end users, exporters and purchasers of the high quality standards of Ontario corn.
  • Accurately determining the levels of DON along with its variants in grain and feed will improve the assessment of the impact of high-DON corn in ethanol production and secondary products such as DDGs, which will benefit livestock producers and provide production efficiencies while reducing human/animal health consequences.
  • The identification of improved BMPs such as fungicides, fungicide timing, and sprayer equipment will benefit farmers through improved return on investment, yields and grain trading opportunities.
  • The development of an effective DON sampling and testing protocol for determining an accurate DON content of a load of corn; this provides a more reliable and efficient grain trade that can be trusted by all parties.
  • The development of a high-capacity grinder and improved DON testing capacity at the retail and end-user facilities will enable effective and efficient implementation of the new DON sampling protocols, which will ensure reliable DON contamination specs in the marketing of corn.
  • The Ontario Grains Researcher Forum kickstarted new collaborations and research project concepts that will continue to drive reductions in the risk of DON in the Ontario corn value chain.

Scientific Summary:

The development of effective management strategies against Gibberella ear rot and the production of mycotoxins is a high priority for the corn industry in the Great Lakes Region.  Economic losses can be significant not only to individual farmers, but the entire corn value chain, especially in epidemic years such as 2018. The 2018 DON crisis in Ontario is estimated to have resulted in a $200 million revenue loss to farmers alone. Several research studies were undertaken following Ontario’s 2018 corn DON epidemic to identify opportunities, from crop inputs through to end use grain handling, that could reduce the risk and impact of another major DON epidemic in Ontario. A summary of outcomes of each of the major objectives is outlined below.

Objective 1: Evaluate corn hybrid resistance to Gibberella ear rot (GER) and DON accumulation and identify mycotoxin dynamics across hybrids and environments.

  • Hybrid differences in DON accumulation have been observed as an opportunity to manage DON in corn production, but growers have no reliable resource for selecting hybrids that may be less susceptible to DON accumulation. To test hybrid differences in DON accumulation, approximately 60 commercial hybrids were entered into misted disease nurseries in the Ontario Corn Committee (OCC) DON trials from 2019 through 2021. Some hybrids were not available every year of the project; 33 hybrids were consistent between 2019 and 2021. It was found that hybrid resistance differences or similarities were not consistent from one planting date to the next across all years of trials. Thus, the resistance screening protocol was improved each year as it was identified that DON resistance ratings were highly linked to weather at silking for each hybrid (and silking date varied notably across the hybrids tested, complicating hybrid comparisons). Warm (27°C – 28°C), humid, rainy days were identified as ideal conditions for infection during corn silking. To capture the influence of the silking date and weather interaction, three planting dates were implemented into the design in 2020 to increase the likelihood of silking during the right weather conditions so that hybrid resistance could be compared more reliably. The research to date indicates that hybrid susceptibility to DON accumulation in current resistance screening protocols is strongly linked to specific weather conditions at silking for each hybrid, which continues to result in a lack of agreement among hybrid resistance data across different locations and years.  The OCC continues to work on a more reliable screening protocol to compare hybrid tolerance to GER and DON accumulation. These data also support the recommendation to plant multiple hybrids with different silking dates to help spread the risk of a whole corn crop facing favorable environmental conditions for GER and high DON accumulation.
  • DON is the most important mycotoxin in Ontario grain corn, but it has a number of similar molecular variants that are becoming more important to the ethanol and feed industry and are not necessarily measured by current DON test kits. DON is converted to DON-3-glucoside (DON3G) as a plant response to de-toxify the DON, resulting in some DON being hidden from detection by current ELISA DON testing methods.  DON3G, when consumed by pigs, converts back to DON in the pig’s gut and therefore can be at toxic concentrations once again. Of the DON-related compounds, DON3G was second only to DON in abundance and could contribute greatly to the total DON-related compounds in a sample, and this is independent of hybrid, especially in cases where DON levels are high.  Another DON-related compound, 15-ADON, contributes relatively little to the total DON in a grain sample at harvest. It is impossible to tell whether the effect was genetic or environmental, as hybrids may silk at different times and infection happens during a narrow window and requires very specific environmental conditions during that time. Research indicated that DON3G concentrations were independent of hybrid, especially in instances of high DON; however, in laboratory testing, DON3G was found to be second in concentration to DON and could contribute significantly to DON-related compounds in a sample. Hybrid did have an effect on 15-ADON in two of the three years of the study, but overall, the concentrations of 15-ADON relative to DON and DON3G were so small that hybrid differences were insignificant.
  • The researchers hypothesised that stressed plants, regardless of hybrid, were likely to be more susceptible to Fusarium infection and have increased DON concentrations at harvest. In plots where plant populations were very high (crowded and therefore stressed), significantly higher concentrations of DON were found. The lowest concentrations of DON, DON3G and total DON were found in corn harvested from low plant density areas (44,400 plants/ha), which differed significantly from the high plant density (113,600 plants/ha). Plant densities of 79,000, 88,900 and 98,800 plants/ha did not differ from the high nor low plant densities for DON, DON3G and total DON accumulation. These results suggest that plants under crowding stress are more susceptible to GER and DON accumulation.
  • Relative emergence of corn plants was also examined for its impact on GER susceptibility. Plants within fields that have delayed emergence usually are stressed through competition from their more advanced neighbouring-adjacent plants. These later stressed plants were hypothesized to be more susceptible to DON accumulation than their earlier-emerged neighbours. The later the delay in plant emergence, the more stress they experience relative to their early emerging counterparts. These delayed stressed plants were found to be more susceptible to DON accumulation, resulting in significantly higher DON concentrations (4- to 9-fold higher) in the grain compared with non-delayed plants. The research indicated that fields with very uneven emergence or plant-plant variability have more stressed plants that are more susceptible to DON accumulation.   
  • The non-Bt hybrid partner in integrated refuge seed mixes was hypothesized as potentially more susceptible for accumulating DON than the primary Bt-hybrid seed in the mix. The refuge portion of integrated refuge seed is selected by seed companies to closely match maturity (flowering and moisture) and have defensive traits like standability and not taller than the Bt-hybrid. The goal of the non-Bt refuge is to reduce the potential development of insect populations resistant to the Bt insecticidal protein(s). There may be few non-Bt hybrid components, so one refuge non-Bt component may be integrated by seed companies across several different hybrids, and these non-Bt components may change from year to year in the same hybrid. Differences were identified in the DON accumulation of the Bt hybrid and its integrated refuge partner hybrid.  In some cases, the Bt hybrid was more susceptible and in other cases it was reversed. Data for DON were pooled across years as there was no interaction between treatment/hybrid and year. The data suggest no difference in DON accumulation among the four hybrids used in this trial, and no difference between the Bt pairs and the refuge of these hybrids; however, we should be careful in making strong conclusions due to the small sample of growing seasons and low disease severity in the sample.
  • With the increase in DON variants and other mycotoxins that could impact the marketability and movement of Ontario grain, the 2019 and 2020 OMAFRA Provincial corn ear mould and mycotoxin survey was expanded to capture not only DON, but the full mycotoxin profile across the province. In 2020, 89% of the samples tested low (< 2 parts per million (ppm)), slightly higher than 2019, but better than 2018 where 40% of samples tested above 2 ppm for DON. The corn samples from 2019 and 2020 were also tested using LC-MSMS to determine the full mycotoxin profile (consisting of 30 mycotoxins). This information is used for short term annual assessments and as a benchmark for future comparisons of mycotoxin profile changes over time. The full mycotoxin profile of the 2019 and 2020 provincial DON survey data found that DON continues to be the most significant mycotoxin; however, DON variants (DON3G, 15ADON and 15ADON3G) are also worth quantifying to more accurately assess overall DON exposure to livestock. No other significant mycotoxins were identified in the province, suggesting that continuing the focus on DON and its derivatives remains appropriate. The NX series of toxins show up in ON at low levels currently and will continue to be monitored.

Objective 2: Develop in-season best management practices (BMPs) for DON mitigation.

  • Research in the past 10 years has shown the best fungicides can reduce mycotoxins by only around 50% using the best application technologies at the right timing of application. Theoretically, there should be a positive relationship between fungicide efficacy and the amount of active ingredient applied to infection channels of the plants (i.e., silks), with some expected differences across various spray delivery systems. At Ridgetown Campus in 2019 and 2022, 9 sprayer systems were evaluated in their effectiveness to deliver a chemical tracer (copper sulphate) to corn silks. The sprayer systems tested included drones (2022 only), aerial (airplane and helicopter), and ground rigs (with various boom and nozzle configurations including drop nozzles) and were ranked for their effectiveness. Results were as follows:
    • The best chemical coverage or dose was produced from the Yield Center 360 Undercover drop pipes, TeeJet Duo nozzles on drop pipes (2019 test only) and the Beluga drop pipes (2022 test only). 
    • The chemical coverage on papers near silks from ground sprayers with overhead broadcast nozzles was approximately 30% less than the coverage from the two drop pipe nozzle systems tested.
    • Chemical coverage from Guardian nozzles were among the lowest of broadcast nozzle configurations, and no different than the flat fan nozzle in 2022. 
    • The chemical coverage from overhead TTI Induction were generally better than the Guardian nozzles, but inferior to any drop pipe configuration.
    • The chemical coverage with the DJI drone was similar to the airplane, and generally inferior to any ground sprayer broadcast sprayers. The Helio drone performed poorly, but it is suspected that the fly pattern was not directly over the water-sensitive papers.  More testing is needed.
    • The helicopter had the lowest coverage and amount of chemical to the papers in 2019 but was the best aerial applicator in 2022.  The huge difference of the helicopter between years is concerning, and thus data needs to be interpreted with caution. 
    • The DJI drone performed very comparably to the plane. More testing is needed.
    • The use of a chemical tracer is incredibly important for evaluating target coverage across sprayer systems.
  • Selection of effective fungicides are key to combating GER and DON accumulation in corn, so several products and timings were evaluated for their efficacy in reducing GER and DON accumulation. Fungicide trial results illustrated some newer foliar fungicide products with multiple active ingredients or modes of action (2- or 3-way formulations) containing prothioconazole, metconazole, or pydiflumetofen were most effective at reducing DON and other foliar diseases. A fungicide product containing a novel active ingredient (pydiflumetofen) was evaluated against conventional triazole fungicides. Efficacy of this fungicide product against GER was similar to the registered triazoles for GER suppression and therefore should provide another mode of action, which should delay the onset of fungicide resistance. This information supports the inclusion of these products in an integrated foliar and ear rot disease management program at the VT/R1 green silk application timing. During the 2 years of this study and across the 5 trials there was no indication that strobilurin fungicides (FRAC Group 11) increase DON production in corn as is the case in wheat. Fungicide timing trials (PreTassel vs Tassel/Silking) showed that the two application timings did not impact disease control or DON accumulation; as expected, fungicide selection had a greater impact.

Objective 3: Develop sampling and testing BMPs for DON.

  • The 2018 DON epidemic was marked by difficulties obtaining consistent, reliable assessments of DON levels in loads of grain delivered to points of sale. Comprehensive research activities were undertaken to identify the sources of this DON sampling and testing variation and to develop new protocols that would improve the reliability of DON results for loads of grain. The research concluded that the main sources of variation in DON testing of loads of grain corn, from greatest to least variability are:

1. Sub-samples of grain from the ~2-kg probed sample (too small)
2. Grinding process
3. Test kit operator
4. Extraction amount
5. Test kit
6. Probe samples

  • The industry method of subsampling a 2 kg probed sample into a 200-500 g sample for grinding, followed by taking 10 or 20 g (depending on the kit) was found to result in the largest source of sampling error and test result inconsistency. The research found that the DON concentration of the 2 kg probe sample, if the entire sample was ground and mixed, was highly representative of the actual DON concentration of the truck load at the elevator. Thus, to minimize the sub-sample source of error, grinding the whole 2-kg probe sample was found to be most effective, followed by a subsample of the ground meal for the test kit.
  • Commercial rapid lateral flow DON test devices were also evaluated for the detection of DON and their cross-reactivity to DON-related compounds. The research team evaluated kits on 50 truckloads, and more than 81 grain handlers and end users also participated in evaluating the kits. Results showed that test kits gave very accurate and repeatable results; however, there was some variability in results depending on the operator, which could be resolved with training. However, laboratory testing also indicated that some commercial test kits overestimated DON concentrations. The results suggested that both Envirologix and Neogen commercial test kits are cross reacting with other DON-related compounds (such as 15ADON) producing results 20-30% higher than the gold standard of LC-MS/MS testing for DON. In conclusion, all test kits were accurate in measuring actual DON. Under the best conditions all the test kits were accurate within 25% of the true answer, which is simply the nature of testing and sampling with mycotoxins. However, the rapid methods (LFDs and ELISA) studied also detected other DON-related compounds (e.g., DON3G) at different amounts depending on each compound’s affinity to the DON-antibody.
  • A decade ago, DON testing relied on ELISA and fluorometric methods and, although accurate at measuring DON in cornmeal, they can cross-react with other DON-related compounds. LC-MS/MS can now be used and is capable of analyzing more than 30 mycotoxins simultaneously, but it is expensive for routine use. Questions existed about the mycotoxins and variants produced by Fusarium graminearum in Ontario corn and their fate during the processing of corn for ethanol production. Similar to results in 2009 research, DON accumulated around 3x in the final dried distiller’s grains with solubles product (DDGS); however, testing only accounted for 78% of DON entering the system, as opposed to the 87% reported in 2009. DON-3-Glc is not concentrated into DDGS, indicating that some DON may have been hydrolyzed into free DON during the fermentation process, confirming early speculation of this occurring in a small subset of samples. The zearalenone mycotoxin, also produced by F. graminearum, was also concentrated in the final DDGS product (2x) but at a lower rate than DON. Swine are the most sensitive domestic species to zearalenone, affecting reproduction.
  • In summary, to address these sources of variation in DON-testing, the research recommends a new sampling protocol for DON testing as follows:
    • A 2 kg sample of corn is required, from 4 probes taken vertically through the grain from the top, 2 from the front and 2 from the back of the lot. 
    • Grind the 2-kg sample to uniform particle size, with 95% passing through a 20-mesh screen (0.841 mm), and thoroughly mix it before a subsample is taken to run a DON test. 
    • All grain receivers that test for DON should have their testing protocol calibrated and validated before receiving the new crop and several times throughout the receiving period.

Objective 4: Identify BMPs for effective storage management of high-DON corn – a case study with grain bags.

  • Grain bags were evaluated for their effectiveness at maintaining corn quality over an extended period, as well as the practicality of using them as a storage system on-farm. At four different sites between January and July 2019, the carbon dioxide levels, oxygen levels, and temperature of four large grain bags were measured on a weekly basis. Grain moisture, water activity, test weight, and mycotoxin content were measured at the beginning of the monitoring period and when the bags were emptied.  Mycotoxin and moisture concentrations were similar for grain sampled at the beginning to those at the end of the period, suggesting that no spoilage occurred, and the grain was safely stored. A steady rise in carbon dioxide, along with a steady decline in oxygen, indicated anaerobic conditions were maintained throughout. The only significant spoilage occurred when the bottom of a bag was perforated by residual crop stubble, allowing water to enter. Overall, however, such spoilage was limited to the perforated areas. The research concluded that grain bags are a useful tool to store excess grain corn in a crisis, provided the grain is dried before storage (<15.5% moisture) and the bags have no perforations. Bags should be placed on well prepared ground that is slightly sloped, with no dips for good drainage, free of any sharp stones and debris, and does not have any stubble from previous crops to ensure the bags remain sealed until unloading.

Objective 5: Coordinate a grinder pilot project to effectively carry out the recommended sampling protocol for DON-infested grain.

  • The new recommended DON sampling and testing BMP requires a 2 kg grain sample to be ground to where 95% can pass through a 20-mesh screen (0.841 mm). No grinders were found at a reasonable price that could grind this amount of whole grain corn quickly enough at that level of fineness in a commercial elevator setting.  A grinder from China was purchased and adapted to suit Canadian standards as a test and demonstration unit. This grinder was re-engineered, and five prototypes were commissioned and sent to grain elevators to test their performance under commercial conditions, all five of the grinders performed similarly and consistently. When using the custom grinders for sample preparation, there was no significant difference in DON concentration between elevator sites and the same samples analyzed in a laboratory setting. The grinder design has since been re-engineered with commercial collaborators in Canada to further improve its performance and efforts are underway to get the improved grinder to market.
  • Further testing was conducted to address the question of cross contamination of high DON corn samples, especially in cases where a high DON sample is followed by a low DON sample.  In cases where samples of about 5 ppm or less were followed by samples of < 1 ppm, the only cleaning required between samples was vacuuming out the top and bottom ports of the grinder while the grinder was running.  Results indicated that the grinder should be opened and cleaned with brush and vacuum following samples that read 10 ppm orgreater. The newer grinder design is expected to have an even simpler clean-out process.

Objective 6: Host an Ontario Grains Researcher Forum to identify research gaps and collaboration opportunities.

  • The Ontario Grains Researcher Forum was a success with about 70 attendees (field crop sector leaders and researchers from across Ontario working on DON and other emerging plant health risk areas and opportunities). Goals of the Forum were considered to be achieved, including the identification of research gaps, multiple networking opportunities, and the development of new collaborations.

Objective 7: Knowledge transfer of project findings and recommendations.

  • Please see project related publications section below for the complete list of knowledge transfer publications related to this project.

External Funding Partners:

This project was funded in part by the Canadian Agricultural Partnership, a five-year federal-provincial-territorial initiative through the Ontario Ministry of Agriculture, Food and Rural Affairs.

Project Related Publications:

Mouldy corn could return in 2019, expert warns. Louis Pin. The Sarnia Observer Newspaper. January 11, 2019.

Preventing DON in Corn: Best Management Tips for 2019. Rachel Telford. Ontario Grain Farmer Magazine. March 2019.

DON corn testing: A new era begins. John Greig. Farmtario. March 11, 2019.

Ontario Corn Committee hybrid DON testing 2018. Dave Hooker, Art Schaafsma and Albert Tenuta. Ontario Corn Committee Release. GoCorn.net. www.FieldCropNews.com. March 21, 2019.

Tips to avoid a repeat of last years DON woes! Conner Lynch. Farmers Forum. May 3, 2019.

Corn School: Fungicide timing critical in highly variable crop. RealAgriculture.com. June 26, 2019.

Focusing on DON: Mycotoxin prevention and management research. Rachel Telford. Ontario Grain Farmers Magazine. February 2020.

Corn School: Evolving fungicides deliver stronger ROI. Albert Tenuta and Kiersten Wise. RealAgriculture.com. March 19, 2020.

Disease Outlook for the 2020 Crop season. Ralph Pearce. Country Guide. April 13, 2020.

Soybean and corn disease threats in Eastern Canada. Treena Hein. Country Guide. May 11, 2020.

Corn yield loss estimates due to diseases in the United States and Ontario, Canada, from 2016 to 2019. Plant Health Progress, American Phytopathological Society. August 20, 2020.

Ontario Diagnostic Day 6: Hitting your target with sprayer applications and getting the most out of fungicides. Jason Deveau, Dave Hooker and Albert Tenuta. RealAgriculture.com. September 23, 2020.

2020 Ontario Grain Corn Ear Mould and Deoxynivalenol (DON) Mycotoxin Survey. Ben Rosser and Albert Tenuta. www.FieldCropNews.com. October 13, 2020.

Average DON levels mean good quality corn year. Farmtario. October 13, 2020.

DON in corn similar to last year. Ontario Farmer. October 14, 2020.

Field observations – 2020 in review. Marty Vermey and Laura Ferrier. Ontario Grain Farmer Magazine. December 2020.

2020 OMAFRA corn ear mould/DON survey results in. Top Crop Manager. October 19, 2020.

Ontario DON levels close to average. Jackie Clark. www.Farms.com. October 29, 2020.

OMAFRA Report: Ministry, farmer organizations complete annual Ontario corn ear mould and DON mycotoxin survey. The Wellington Advertiser newspaper. December 30, 2020.

OMAFRA 2020 Corn Seasonal Summary. Ontario Farmer Newspaper and www.FieldCropNews.com. December 30, 2020.

DON-infected corn – Learnings from two research projects. Ontario Grain Farmer Magazine. August 2021.

2021 Ontario Grain Corn Ear Mould Survey. Tenuta and Rosser. www.FieldCropNews.com. October 25, 2021.

DON in corn – What to look for. Ontario Grain Farmer Magazine. October 2021.

Testing for DON in four key steps. Ontario Grain Farmer Magazine. October 2021.