Location: Mycotoxin Prevention and Applied Microbiology Research
2025 Annual Report
Objectives
Objective 1: Define diversity of mycotoxin-producing Fusarium species. [C1, PS1, PS2]
Sub-objective 1.A: Elucidate phylogenetic diversity, mycotoxin potential, and pathogenicity to cereals of fungi in the F. tricinctum species complex.
Sub-objective 1.B: Identify genomic and phenotypic differences in collections of F. proliferatum and F. verticillioides isolates to aid discovery of targets for control of fumonisins in corn.
Objective 2: Identify targets to reduce fumonisin contamination in corn. [C1, PS1, PS2, PS5]
Sub-objective 2.A: Determine whether the corn zmCRR1 protein contributes to resistance to fumonisin contamination.
Sub-objective 2.B: Identify corn genes encoding papain-like cysteine proteases involved in fumonisin contamination to aid genomics-assisted breeding.
Sub-objective 2.C: Reduce fumonisin contamination in corn by engineering kernel-specific expression of RNAi targeting the fumonisin biosynthetic gene FUM1.
Sub-objective 2.D: Determine how corn oxylipins control fumonisin production in F. verticillioides.
Sub-objective 2.E: Determine whether the killer meiotic drive element SkK can be used to drive biased transmission of a gene that blocks fumonisin production in F. verticillioides.
Approach
Fusarium species are fungi with potentially the greatest negative impact on agriculture. This is because of their collective abilities to produce mycotoxins and cause destructive diseases in crops, including the important cereals: corn, wheat, and rice. The Fusarium mycotoxins fumonisins and trichothecenes are among the mycotoxins of most concern to food and feed safety due to their toxicity and frequent occurrence in crops. However, Fusarium species produce other mycotoxins whose effects on food and feed safety are poorly understood. In the U.S., harmful impacts of mycotoxins on health are mitigated by removing contaminated grain from food/feed supply chains. Despite these efforts, however, the toxins continue to cause billions of dollars in agricultural losses. This project plan addresses knowledge gaps that hinder control of mycotoxins caused by two groups of Fusarium: the Fusarium tricinctum species complex (FTSC), which includes multiple species that cause head blight of small-grain cereals and produce multiple mycotoxins; and the F. fujikuroi species complex, specifically Fusarium proliferatum and Fusarium verticillioides, which are the primary causes of fumonisin contamination in corn. The proposed research has two objectives: i) define diversity of mycotoxin-producing Fusarium species, specifically members of the FTSC, F. proliferatum, and F. verticillioides; and ii) identify targets to reduce fumonisins in corn. To address the first objective, we propose to elucidate variation in genome sequences, mycotoxin production, and pathogenicity within and among Fusarium species. This will aid development of broadly effective control practices for Fusarium mycotoxins. To address the second objective, we propose to identify corn and Fusarium proteins/genes that can be used to enhance breeding or engineering strategies aimed at reducing fumonisin contamination. To address the second objective, we also propose to develop fumonisin reduction methods based on two biological phenomena: RNA interference and meiotic drive elements. The research accomplishments will aid efforts to reduce mycotoxin contamination in corn and other cereal crops, and will benefit growers, processors, regulatory agencies, and ultimately American consumers.
Progress Report
Objective 1: Mycotoxins are dangerous chemicals produced by some fungi. Fumonisins are among the mycotoxins of most concern to food and feed safety because of their toxicity and frequent occurrence in crops, particularly corn. We and ARS scientists in Ithaca, New York, searched publicly available databases of genome sequences of thousands of fungi to determine which fungi have the genetic potential to produce fumonisins. The results revealed that the genetic potential for fumonisin production is limited to relatively few species of five fungi: Alternaria, Aspergillus, Bipolaris, Tolypocladium, and Fusarium. These species include fungi that cause crop or insect diseases, or that occur in the soil or on decaying plants. Chemical analyses of some of the species confirmed that they produce fumonisins or structurally similar toxins known as AAL toxins. The results also indicate that the number of species that produce fumonisins has expanded by direct transfer of fumonisin production genes between species. These findings have identified the sources of fumonisin contamination in different environments. Knowing the sources, will focus and improve efficiency of efforts to reduce fumonisin contamination.
The fungi Fusarium proliferatum and Fusarium verticillioides are the predominant causes of fumonisin contamination in corn, but it is unclear how genetic differences between and within the species contribute to variation in fumonisin contamination. To address this knowledge gap, we compared the genome sequences generated from a worldwide collection of the two species. The results indicate that genetic variation is greater among isolates of F. proliferatum than F. verticillioides. This suggests F. proliferatum has greater potential to adapt to changing agricultural practices. The results also indicate that each species consists of four genetically distinct groups and revealed that some groups are more prevalent than others in certain regions of the world. For example, only two of the F. verticillioides groups were detected in the United States and Canada, but all four groups were detected in Mexico and Guatemala. In addition, 4% of isolates of both species had genetic mutations that impair fumonisin production. This improved understanding of genetic differences between and within F. proliferatum and F. verticillioides will enhance efforts to reduce fumonisin contamination. This in turn will improve safety of corn and reduce economic losses to farmers caused by the contamination.
Computer models that predict mycotoxin contamination in crops can help farmers choose agricultural practices that prevent or reduce contamination and the resulting financial losses. Therefore, we conducted research to increase the accuracy of predictive models for mycotoxin contamination in Illinois corn. To this end, we investigated the occurrence of mycotoxin-producing fungi in 40 agricultural fields in 21 counties across Illinois. Our goal was to determine how conditions before planting, during the growing season, and after harvest impact levels of two mycotoxins (fumonisins and aflatoxins) in corn harvested from the same fields. Fumonisin levels were low across Illinois, but the levels in the south were higher than in central or northern Illinois. The levels of another mycotoxin, aflatoxin, were low and similar across Illinois. Identification of fungi recovered from the 40 fields are in progress, but to date they indicate very few isolates of aflatoxin producing fungi, which explains the low level of aflatoxin contamination. Incorporation of information obtained in this research into predictive models will improve the models and thereby minimize mycotoxin levels in Illinois corn and the negative economic impact that contamination has on farmers.
Fumonisin contamination of corn is often higher in southern Illinois than central and northern regions of the state. To investigate the cause(s) of this difference, we determined the composition of Fusarium species in corn harvested from across the state in 2021 and 2023. Using morphological and DNA-based methods, we identified seven species of Fusarium that occur in the corn. Notably, the fumonisin-producing species F. verticillioides was found more frequently in southern Illinois than other areas of the state. These findings provided evidence that the higher levels of fumonisin contamination in southern Illinois are likely caused by a higher incidence of F. verticillioides rather than solely by environmental conditions that favor fumonisin production by the fungus. This information will help farmers in southern Illinois to reduce fumonisin contamination in the future by selecting management strategies that target F. verticillioides.
Objective 2: Enhancing resistance of corn to diseases caused by the fungus Fusarium is a highly effective way to reduce economic losses to farmers caused by the diseases and their associated mycotoxin contamination problems. A key step to enhance disease resistance is identification of proteins produced by corn and Fusarium that impact resistance and then use the resulting information to develop control strategies; for example, by selecting corn varieties that have proteins that are most effective at combatting Fusarium. To this end, we investigated how Fusarium proteins inhibit a corn defense protein known as papain-like cysteine protease (PLCP). As a first step in this process, we developed two experimental protocols to identify the inhibitory proteins produced by Fusarium. The second protocol identified a group of about 24 Fusarium proteins, one or more of which inhibits the corn PLCP protein by degrading it. In future experiments, we plan to identify which of the Fusarium proteins is responsible for the degradation and whether changes in the amino acid sequence of the PLCP protein make it resistant to degradation by the Fusarium protein. This research has potential to identify novel mechanisms that enhance resistance of corn to Fusarium-incited diseases and mycotoxin contamination.
In collaboration with ARS in Ames, Iowa, we developed three online databases to aid identification of additional corn proteins that enhance resistance of this important crop to diseases and mycotoxin contamination caused by the fungus Fusarium. To do this, we utilized artificial intelligence (AI)-assisted computer modeling systems and integrated the information into an online database of corn genetic resources known as MaizeGDB. The three Fusarium online databases and their uses are as follows: 1) the Fusarium Protein Toolkit provides information on amino acid sequences of all proteins produced by six Fusarium species and enables database users to investigate protein functions; 2) the PanEffect database allows users to investigate how variation in amino acid sequences of Fusarium proteins impacts their functions and their interaction with corn proteins; 3) the SNPversity Tool allows users to investigate genetic variation in both Fusarium and corn and to assess correlations between genetic variation and geographic origin of corn varieties. Over the past year, the databases have been visited over 12,000 times, which is a testament to their growing importance to research aimed at enhancing resistance of corn to Fusarium-incited diseases and mycotoxin contamination. This research has excellent potential to provide farmers with additional methods to reduce economic losses caused by Fusarium and to improve the quality and safety of corn produced in the United States.
We are also developing corn lines that are resistant to Fusarium infection using a strategy known as RNA interference (RNAi). In cooperation with university researchers in Madison, Wisconsin, we generated eight genetically modified corn lines that produce small RNAi molecules that prevent formation of a Fusarium enzyme (Fum1) that is required for fumonisin production. We anticipate that if the modified corn can block formation of Fum1, it will reduce fumonisin contamination of corn. We have developed protocols to confirm the modification has stably integrated into the corn genome and plan to test whether it prevents fumonisin contamination. We have also established a formal agreement with the company Beck’s Hybrid, which plans to incorporate the modified corn into its breeding program aimed at reducing mycotoxin contamination in corn.
Some strains of the fungus Trichoderma and the bacterium Bacillus can protect crops from plant pathogenic microbes; but it is unclear whether they can protect corn from Fusarium-incited diseases and mycotoxin contamination. To fill this knowledge gap, we investigated whether the Trichoderma and/or Bacillus strains could also protect corn from fumonisin contamination caused by the fungus Fusarium verticillioides. To do this, we obtained corn seed treated with either Trichoderma or Bacillus strains, planted the corn in experimental field plots, and then infected the corn with F. verticillioides. In our first field trial, treatment with Trichoderma or Bacillus did not impact fumonisin contamination; but we are repeating the study in 2025 to confirm the results. This research has the potential to provide farmers with an additional method to reduce economic losses caused by fumonisin contamination of corn.
Accomplishments
1. A protein database to aid control of crop diseases and toxin contamination. The fungus Fusarium causes billions of dollars in losses to world agriculture annually by causing destructive crop diseases and contaminating infected crops with toxins that are health hazards to humans, livestock and pets. Each Fusarium species has 10,000 genes that serve as blueprints for proteins, each with an important role in one or more biological processes. Little is known about which proteins produced by Fusarium species contribute to their ability to cause crop diseases and toxin contamination, but such proteins can be critical for development of more effective strategies to control Fusarium-incited diseases and toxin contamination. To address this significant knowledge gap, ARS scientists in Peoria, Illinois, and Ames, Iowa, developed the Fusarium Protein Toolkit, an interactive and artificial intelligence-assisted online database that includes computer-generated models for all proteins produced by 22 Fusarium species. The interactive nature of the database allows users to investigate predicted structures and functions of the proteins, including how they contribute to the ability of Fusarium to infect crops. The database also allows users to investigate whether small differences in the same protein can impact function. The database is being used to identify control strategies that reduce crop diseases and toxin contamination caused by Fusarium. Evidence of the tremendous utility of the database is that, over the past 9 months, the Fusarium Protein Toolkit has been accessed over 6,000 times by 360 different users.
2. Toxin-contaminated corn can serve as a safe food source for beneficial insects. Fumonisins are among the fungal toxins of most concern to food and feed safety because of their frequent occurrence in corn. Contamination of kernels with the toxins reduces the value and utility of corn, resulting in agricultural waste and lost income for farmers. To avoid such financial losses, ARS scientists in Peoria, Illinois, and Stoneville, Mississippi, determined whether it was possible to use fumonisin-contaminated corn to produce yellow mealworms, insect larva that are being evaluated as a nutrition supplement for livestock feed. The scientists found that when fed fumonisin-contaminated corn kernels, yellow mealworms exhibited no negative effects on growth or survival, and they retained only 0.5% or less of the fumonisin that was in the corn. These findings indicate that yellow mealworms can be safely produced on fumonisin-contaminated corn for application as a livestock feed supplement, creating a new market for previously wasted corn and improved revenue for farmers with contaminated corn.
3. Soil bacteria protect crop plants by not triggering the plant immune system. Some bacteria live in the soil near plant roots without causing disease. There is growing evidence that these bacteria can protect crops from fungi that cause diseases and toxin contamination and, therefore, have potential as a pest management tool. For such bacteria to be an effective management tool, they must be able to grow in soil near roots without triggering the plant immune system. But how the bacteria do this is unclear. To address this knowledge gap, ARS scientists in Peoria, Illinois, and their university collaborators in Princeton, New Jersey, demonstrated that the bacteria secrete an enzyme that quickly degrades a key protein that triggers the plant immune system. This discovery advances the understanding of how bacteria can live near roots and protect crops from pathogens. This knowledge has potential to give farmers another pest management tool to control fungal diseases and toxin contamination.
4. Identification of emerging disease and toxin contamination problems in corn. Corn is grown worldwide and serves as an essential staple crop. Corn ear rot is among the most destructive diseases of corn because it reduces the quality, quantity, and safety of the grain, which in turn lead to financial losses to farmers and potential hazards to human and animal health. Understanding factors that cause corn ear rot outbreaks in other countries is critical to ensuring that such outbreaks do not occur in the United States. Ethiopia frequently experiences devastating outbreaks of corn ear rot. To better understand which fungi cause these outbreaks, ARS scientists in Peoria, Illinois, and collaborators in Ethiopia surveyed corn-growing regions of Ethiopia in 2020 and 2021 for ear rot. Fungi causing corn ear rot were isolated from rotted kernels and identified using morphological and DNA-based methods. Approximately 25% of the fungi were Fusarium species that are rarely found in the United States. Thus, Fusarium species that are not yet a problem in the United States can cause significant disease and toxin contamination under some environmental conditions. This benefits farmers in the United States by providing information on the potential risks to corn production.
5. Controlling contamination of corn with fungal toxin by identifying chemicals that fungi use to communicate. Together, spoilage, disease, and toxin contamination of crops caused by fungi can result in hundreds of millions of dollars in annual losses to U.S. agriculture. Fungi known as Aspergillus, Fusarium and Penicillium are among the biggest contributors to these losses. These fungi can occur together on the same crop plant, but it is unclear how they affect one another’s ability to cause disease and/or produce toxin, and whether such interactions can affect efforts to reduce toxin contamination. To address this knowledge gap, ARS scientists in Peoria, Illinois, determined how four chemicals produced by Fusarium, Aspergillus and/or Penicillium impact the fungus Fusarium verticillioides, which is a toxin-producing pathogen of corn. The four chemicals caused profound changes in F. verticillioides as measured by changes in gene expression (i.e., the level at which genes are on or off). Some genes were affected in a similar way to all four chemicals, while others were only affected by one or two chemicals. These observations demonstrate that the chemicals are a critical component of mechanisms that F. verticillioides uses to detect and compete with other fungi. Further, some of the genes that are affected by the chemicals are potential targets for control strategies to reduce disease and mycotoxin contamination of corn caused by F. verticillioides. Such reductions will improve the safety of United States corn to humans and livestock and reduce economic losses to farmers caused by diseased and toxin-contaminated corn.
6. Fruiting body color – a potential target to control crop diseases and toxin contamination. Species of the fungus Fusarium cause disease of numerous crops and can contaminate the crops with toxins that are hazardous to humans, pets and livestock. For Fusarium to cause disease it must survive and spread in the environment. To do this, it produces spores in red or dark purple fruiting bodies. While science has long known that the spores are required for spread and survival, the importance of pigmentation of the fruiting bodies is unclear. To fill this knowledge gap, ARS scientists in Peoria, Illinois, and university collaborators in Aalborg, Denmark, characterized the genes required for production of the chemical that causes the red pigmentation of fruiting bodies produced by the species Fusarium vanettenii, which is a pathogen of legume crops. The scientists also determined the chemical structure of the red pigment. These findings provide tools that researchers can use to determine whether fruiting body pigmentation affects spore spread and survival, and whether blocking fruiting body pigmentation is an effective pest management strategy for legumes. If effective, the strategy would have potential application in control of disease and toxin contamination of corn and wheat caused by other Fusarium species.
Review Publications
Temesgen, D., Adugna, G., Suresh, L.M., Bekeko, Z., Iriarte-Broders, G., Vaughan, M.M., Proctor, R., Mehl, H.L., Prasanna, B.M., Opoku, J. 2024. Fusarium boothii, Fusarium meridionale, and Fusarium temperatum are emerging preharvest maize ear rot pathogens in Ethiopia. Plant Disease. https://doi.org/10.1094/PDIS-12-23-2765-SR.
Eastman, S., Jiang, T., Ficco, K., Liao, C., Jones, B., Wen, S., Biddle, Y.O., Eyceoz, A., Yatsishin, I., Naumann, T.A., Conway, J.M. 2024. A type II secreted subtilase from commensal rhizobacteria cleaves immune elicitor peptides and suppresses flg22-induced immune activation. Cell Reports. https://doi.org/10.1016/j.celrep.2024.115063.
Brown, D.W., Kim, H.-S., Proctor, R.H., Wicklow, D.T. 2024. Low molecular weight acids differentially impact Fusarium verticillioides transcription. Fungal Biology. https://doi.org/10.1016/j.funbio.2024.08.007.
Gold, S.E., Brown, D.W., Williams, F.N., Naden, B.D., Vo, V., Miller, C.E. 2024. A Fusarium verticillioides MAT1-2 strain near isogenic to the sequenced FGSC7600 strain for producing homozygous multigene mutants. The Journal of Fungi. 10, 592. https://doi.org/10.3390/jof10080592.
Kim, H., Haley, O., Portwood II, J.L., Harding, S.F., Proctor, R., Woodhouse, M.H., Sen, T.Z., Andorf, C.M. 2024. Fusarium Protein Toolkit: A web-based resource for structural and variant analysis of Fusarium species. BMC Microbiology. https://doi.org/10.1186/s12866-024-03480-5.
Paulk, R.T., Abbas, H.K., Rojas, M.G., Morales Ramos, J.A., Busman, M., Little, N., Shier, W.T. 2024. Evaluating Tenebrio molitor (Coleoptera: Tenebrionidae) for the remediation of fumonisin B1 levels in livestock feed. Journal of Economic Entomology. 118(1), 2025, 63–70.. https://doi.org/10.1093/jee/toae273.