Research Project: Innovative Food and Feed Safety Research to Eliminate Mycotoxin Contamination in Corn and other Crops

Location: Mycotoxin Prevention and Applied Microbiology Research

2022 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 focuses on understanding the diversity in individual species of Fusarium or groups of species of the fungus. One group is the Fusarium tricinctum species complex (FTSC), which occurs on diverse crop species but is of particular concern as a cause of Fusarium head blight, a disease of wheat and barley that occurs in many regions of the world where these crops are grown. We have examined a global collection of isolates of the FTSC recovered from diverse crops to develop an assay that can distinguish between individual species within FTSC. The assay is based on variation in the DNA sequence of a gene involved in phosphate transport. This research provides information that will help plant pathologists, plant breeders, and regulatory organizations to detect and manage crop diseases and toxin contamination caused by the FTSC. We are also determining the extent of genetic diversity in two Fusarium species, F. proliferatum and F. verticillioides, that are the primary causes of contamination of crops with fumonisin toxins, which is one of the fungal toxins of most concern to food and feed safety. Despite the importance of these fungi to agriculture, there is a knowledge gap as to whether strains from different geographic regions and/or crops vary in genetic diversity, fumonisin production, and ability to cause crop diseases. Therefore, we are assembling a worldwide collection of isolates of the two species to assess variation in genetic diversity, fumonisin production, and ability to cause disease of corn. To assess genetic diversity, we have generated whole genome sequence data for 55 – 60 strains of each species and shown that the level of diversity in genome sequences of F. proliferatum is markedly higher than in F. verticillioides. Knowledge of diversity that exists within F. proliferatum and F. verticillioides will aid in developing robust agricultural practices to reduce fumonisin contamination in corn and other crops. In collaboration with researchers at Agriculture and Agri-Food Canada, we also elucidated the genetic diversity and differences in toxin production in the Fusarium buharicum species complex (FBSC), another group of Fusarium species that are poorly characterized. The diversity analysis indicates that the FBSC consists of seven genetically distinct species. Analysis of whole genome sequences from each species indicated that one species has the genes necessary for production of trichothecene toxins and another species has genes necessary for fumonisin toxins. These findings are significant because trichothecenes and fumonisins are among the fungal toxins of most concern to food and feed safety. In chemical analyses of the species, trichothecenes were detected but fumonisins were not. This research provides knowledge of the genetic diversity of fungi in the FBSC as well as the potential of members of the complex to cause toxin contamination in crops. Thus, the research also provides information on the risk that the FBSC poses to food and feed safety. Genome sequence data for diverse species of the fungus Fusarium have been generated by research organizations around the world and are being used to identify and develop strategies to control diseases and toxin contamination of crops. However, most Fusarium genome sequence data is of moderate quality, and this limits their usefulness. Therefore, in collaboration with researchers at the University of Massachusetts and the U.S. Department of Energy’s Joint Genome Institute, we are generating high-quality genome sequence data for 45 species that represent the breadth of genetic diversity of Fusarium. The high-quality genome sequence data consist of DNA sequences corresponding to full-length chromosomes and facilitate analyses that are not possible with most of the data that has existed until now. This in turn will enhance research efforts aimed at identifying strategies to control Fusarium-incited crop diseases and toxin contamination problems. Objective 2 of the project is to identify targets to reduce fumonisin contamination in corn. One approach we are using to meet this objective is to identify enzymes that corn produces to defend itself from disease-causing fungi as well as enzymes that the fungi produce to overcome corn defenses. Knowledge of these enzymes and how they interact will be used to strengthen corn’s defenses against Fusarium and to reduce toxin contamination in the field. During the current year, we discovered that the plant defense protein CRR1 disrupts development of fungi by causing their spores to clump together. Because CRR1 was first identified in cotton, we had to use a DNA-sequence approach to identify the corn and wheat versions of CRR1. We are currently purifying the corn and wheat proteins to confirm that they have the same function as the CRR1 protein from cotton. We also discovered that some fungi, including the toxin-producing fungi Fusarium graminearum and Fusarium verticillioides, produce a protein that deactivates CRR1 by cleaving it into two pieces. We have purified the Fusarium proteins, determined their amino acid sequence, and used the amino acid sequences to identify the genes in the two Fusarium species that serve as blueprints for the CRR1-cleaving proteins. We then used the genes to genetically engineer yeast to produce large quantities of the proteins that can be used for detailed analyses on how the proteins function. These research results provide novel and fundamental knowledge of protein-protein interactions that occur when corn interacts with toxin-producing fungi. This knowledge has potential to guide plant breeding and engineering efforts focused on reducing diseases and toxin contamination of corn and other crops. Another approach used to meet Objective 2 is to assess whether a F. verticillioides gene known as Spore Killer can be used to reduce fumonisin contamination. The Spore Killer gene is classified as a Meiotic drive element (MDE). In general, MDE genes are transmitted at a higher-than-expected frequency from parents to offspring, and as a result can spread rapidly in populations of organisms that undergo sexual reproduction. When a strain of F. verticillioides with Spore Killer mates with a strain that lacks the gene, the gene is transmitted to 100% of offspring rather than the expected 50%. In collaboration with researchers at Illinois State University, we are investigating whether the Spore killer gene can be linked to a gene that suppresses fumonisin production and thereby cause both genes to be spread rapidly in populations of F. verticillioides in corn fields. This is expected to reduce the frequency of fumonisin-producing strains, which in turn should reduce fumonisin contamination in the corn. During the past year, we identified the DNA sequence of the Spore Killer gene and determined that another genetic phenomenon, known as Meiotic Silencing by Unpaired DNA (MSUD), suppresses the function of Spore Killer under some circumstances. This research has furthered the understanding of the Spore Killer gene in F. verticillioides and provides information that is necessary to harness the gene’s function to reduce fumonisin contamination.

Accomplishments
1. Developed high-tech tools to accurately identify toxin-producing fungi. Collectively, species of the fungus Fusarium cause diseases and toxin contamination of most crops. The toxins pose health hazards, and together the diseases and contamination result in multibillion dollar losses to world agriculture each year. One barrier to effectively controlling these agricultural problems is difficulties in identifying and distinguishing between some species of Fusarium that cause the problems. Therefore, ARS researchers in Peoria, Illinois, and researchers at Pennsylvania State University have developed DNA sequence-based tools to accurately identify species of Fusarium (https://github.com/fusariumid/fusariumid). In addition, the researchers have published detailed protocols that describe how to use the tools most effectively. One of the tools is a database of DNA sequences from over 200 Fusarium species. Sequences from newly isolated Fusarium strains from diseased and/or toxin-contaminated crops can be compared to sequences in the database to determine the species identity of the new strains. Thus, the research provides tools that will enable plant pathologists and researchers worldwide to accurately determine the species identity of Fusarium strains that cause agricultural problems. This in turn will allow plant pathologists to select the most appropriate control practice(s) to combat the problems.

2. Identified a spore-killing gene to reduce fungal toxins in crops. The fungus Fusarium produces toxins that contaminate crops, pose health hazards, and reduce the value of U.S. crops by tens of millions of dollars annually. ARS researchers in Peoria, Illinois, and researchers at Illinois State University have identified a gene in a Fusarium species (F. verticillioides) that can spread through populations of the fungus via the fungus’ spore production process (sporulation). The research demonstrated that when F. verticillioides undergoes sporulation, all spores that inherit the gene develop to maturity while spores that do not inherit the gene die. This research filled a long-standing knowledge gap concerning the gene responsible for the spore-killing phenomenon in F. verticillioides, which frequently contaminates corn with cancer-causing toxins known as fumonisins. The new knowledge points to a novel approach to control fumonisin contamination by physically linking the spore-killing gene to a gene that suppresses fumonisin production. Linking the two genes should facilitate spread of the fumonisin suppression gene in local populations of F. verticillioides as strains of the fungus undergo sexual reproduction in agricultural fields. The resulting reduction in fumonisin contamination would prevent losses in the value of corn caused by contamination and would improve the safety of corn for use as human food and animal feed.

3. Analyzed diversity of fungi that cause disease and toxin contamination in crops. Fusarium head blight (FHB) is one of the most destructive diseases of wheat and other cereals worldwide because it reduces yield and quality, and it results in contamination of grain with toxins that pose health hazards. A group of poorly characterized fungi known as the Fusarium tricinctum species complex (FTSC) are an important cause of FHB in many regions of the world, but these species and their individual abilities to produce toxins are poorly understood. Therefore, ARS researchers in Peoria, Illinois, surveyed a collection of 151 FTSC strains isolated from cereals and other crops from around the world to assess the genetic diversity and toxin production of the strains. DNA-sequence based analyses indicated that the strains comprised 24 genetically distinct species, including nine species that are new to science. Collectively, the 24 species produced three toxins that are of minor safety concerns, but production ability differed among some species. Further, none of the species produced trichothecene toxins, which are produced by F. graminearum and among the toxins of most concern to food and feed safety. This research helps to clarify information on fungi associated with FHB and their toxin production abilities. The findings will guide plant breeding and other efforts to develop control practices that reduce FHB and toxin contamination in cereal crops.

4. Improved knowledge of fungal enzymes that contribute to resistance to corn ear rot. Some fungi cause ear rot disease of corn and contaminate infected kernels with toxins. The fungi also produce protease enzymes that inactivate enzymes that corn produces to defend itself from pathogens. But a detailed understanding of how the fungal enzymes function and interact with the corn enzymes is lacking. Determination of the three-dimensional (3D) structure of enzymes is critical for a detailed understanding of how the fungal and corn enzymes function and interact with one another. Therefore, ARS researchers in Peoria, Illinois, developed a method to produce pure fungal proteases in order to determine the 3D structure using X-ray technology. Furthermore, the ARS researchers incorporated the mineral selenium into the production process because the mineral enhances the quality of structural information obtained by the X-ray technology. This research provided a novel method for production of fungal enzymes to facilitate determination of their 3D structure, which will fill a knowledge gap in how the fungal enzymes function and interact with corn defense enzymes. This knowledge will aid plant breeding and engineering efforts to improve resistance of corn to ear rot and toxin contamination caused by fungi.

5. Analyzed molecules responsible for controlling diseases and toxin contamination of crops. The fungus Fusarium causes billions of dollars in losses to world agriculture each year by causing destructive crop diseases and contaminating infected crops with toxins that pose health hazards. Some of the toxins as well as some other metabolites that Fusarium produces to enhance its ability to cause crop diseases belong to a group of molecules known as polyketides, and polyketides are synthesized via the activity of enzymes known as polyketide synthases (PKSs). Given the importance of polyketides in the ability of Fusarium to cause crop disease, PKSs are potential targets to control the diseases. Therefore, ARS researchers in Peoria, Illinois, analyzed the genome sequences of 206 species of Fusarium to identify genes that serve as blueprints for PKSs. The survey revealed that the Fusarium species have the collective ability to produce 113 structurally distinct polyketides. These results provide critical information on two contrasting aspects of Fusarium chemistry: variation in the ability of Fusarium to produce polyketides that pose health hazards (toxins) and polyketides that are targets for control of disease and the associated toxin contamination of crops. Thus, this research contributes to the understanding of risks that diverse Fusarium species pose to food and feed safety, and it contributes to a worldwide effort to identify targets to control crop diseases and toxin contamination caused by Fusarium.

Review Publications
Kim, H.-S., Park, S.-Y., Kang, S., Czymmek, K.L. 2022. Time-lapse imaging of root pathogenesis and fungal proliferation without physically disrupting roots. In: Coleman, J., editor. Fusarium wilt. Methods in Molecular Biology, vol 2391. New York, NY: Humana. p. 153-170. https://doi.org/10.1007/978-1-0716-1795-3_13.
O'Donnell, K., Laraba, I., Geiser, D.M. 2022. Pure culture and DNA sequence-based identification of fusarium from symptomatic plants and diverse substrates. In: Coleman, J., editor. Fusarium Wilt. Methods in Molecular Biology, vol 2391. New York, NY: Humana. p. 1-20.
Nichea, M.J., Proctor, R., Probyn, C.E., Palacios, S.A., Cendoya, E., Sulyok, M., Chulze, S.N., Torres, A.M., Ramirez, M.L. 2021. Fusarium chaquense,sp. nov, a novel type A trichothecene-producing species from native grasses in a wetland ecosystem in Argentina. Mycologia. 114(1):46-62. https://doi.org/10.1080/00275514.2021.1987102.
Dowd, P.F., Naumann, T.A., Johnson, E.T. 2022. A maize gene coding for a chimeric superlectin reduces growth of maize fungal pathogens and insect pests when expressed transgenically in maize callus. Plant Gene. 30. Article 100359. https://doi.org/10.1016/j.plgene.2022.100359.
O'Donnell, K., Whitaker, B.K., Laraba, I., Proctor, R.H., Brown, D.W., Broders, K., Kim, H.-S., McCormick, S.P., Busman, M., Aoki, T., Torres-Cruz, T.J., Geiser, D.M. 2022. DNA sequence-based identification of Fusarium: A work in progress. Plant Disease. 106(6):1597-1609. https://doi.org/10.1094/PDIS-09-21-2035-SR.
Naumann, T.A., Sollenberger, K.G., Hao, G. 2022. Production of selenomethionine labeled polyglycine hydrolases in Pichia pastoris. Protein Expression and Purification. 194. Article 106076. https://doi.org/10.1016/j.pep.2022.106076.
Brown, D.W., Kim, H.-S., McGovern, A.E., Probyn, C.E., Proctor, R.H. 2022. Genus-wide analysis of Fusarium polyketide synthases reveals broad chemical potential. Fungal Genetics and Biology. 160. Article 103696. https://doi.org/10.1016/j.fgb.2022.103696.
Laraba, I., Busman, M., Geiser, D.M., O'Donnell, K. 2022. Phylogenetic diversity and mycotoxin potential of emergent phytopathogens within the Fusarium tricinctum species complex. Phytopathology. 112(6):1284-1298. https://doi.org/10.1094/PHYTO-09-21-0394-R.
Kang, S., Lumactud, R., Li, N., Bell, T.H., Kim, H.-S., Park, S.-Y., Lee, Y.-H. 2021. Harnessing chemical ecology for environment-friendly crop protection. Phytopathology. 111(10):1697-1710. https://doi.org/10.1094/PHYTO-01-21-0035-RVW.
Munkvold, G.P., Proctor, R., Moretti, A. 2021. Mycotoxin production in Fusarium according to contemporary species concepts. Annual Review of Phytopathology. 59(1):373–402. https://doi.org/10.1146/annurev-phyto-020620-102825.
Lohmar, J.M., Rhoades, N.A., Patel, T.N., Proctor, R., Hammond, T.M., Brown, D.W. 2022. A-to-I mRNA editing controls spore death induced by a fungal meiotic drive gene in homologous and heterologous expression systems. Genetics. 221(1). Article iyac029. https://doi.org/10.1093/genetics/iyac029.