Research Project: Genomic and Metabolomic Approaches for Detection and Control of Fusarium, Fumonisins and Other Mycotoxins on Corn

Location: Mycotoxin Prevention and Applied Microbiology Research

2020 Annual Report

Objectives
Objective 1: Use comparative phylogenomic approaches to enable accurate identification of mycotoxigenic Fusarium and to elucidate components of Fusarium genomes that are responsible for variation in mycotoxin production. Sub-objectives 1.1 through 1.3 are as follows: 1.1 – Develop a DNA sequence database that facilitates accurate identification of all toxigenic Fusarium species; 1.2 – Determine whether mycotoxin biosynthetic gene clusters and genetic networks that regulate cluster expression differ in their distributions among Fusarium species; 1.3 – Determine whether F. verticillioides has genes that repress fumonisin production. Objective 2: Develop and utilize liquid chromatography-mass spectrometry (LC-MS) approaches for metabolomic analysis of Fusarium verticillioides infection of maize. Sub-objective 2.1 and 2.2 are as follows: 2.1 – Develop workflows for untargeted analyses of the metabolomes of maize, F. verticillioides, and the maize-F. verticillioides interaction; and 2.2 – Identify metabolic biomarkers for high and low levels of F. verticillioides-induced disease in maize. Objective 3: Identify and characterize plant and fungal factors that can impact mycotoxin contamination via their effects on plant disease development. Sub-Objective 3.1 through 3.4 are as follows: 3.1 – Determine how primary sequence and secondary structure of fungal polyglycine hydrolases affect the inhibitory activity of this class of proteases against plant chitinases; 3.2 – Isolate and identify ChitA alloform-specific proteases secreted by the fungi Stenocarpella maydis and Trichoderma viride; 3.3 – Elucidate the role of plant class IV chitinases in maize-fungus interactions; and 3.4 – Identify candidate receptor and regulatory genes that mediate oxylipin-induced changes in expression of fumonisin biosynthetic genes and fumonisin production in F. verticillioides. Objective 4: Identify and characterize components of fungus-fungus interactions that contribute to or inhibit mycotoxin contamination of crops. Sub-objective 4.1 through 4.3 are as follows: 4.1 – Sample across different climate zones to identify novel fungal endophytes of maize that inhibit growth and/or fumonisin production in F. verticillioides; 4.2 – Identify candidate genes in Talaromyces that are responsible for inhibition of growth in F. verticillioides; and 4.3 – Determine whether production of fumonisins and other mycotoxins contributes to the competitiveness of F. verticillioides with other Fusarium species.

Approach
The fungus Fusarium is of concern to agriculture because it can cause crop diseases and produce mycotoxins, including three (fumonisins, trichothecenes, and zearalenone) that are among the mycotoxins of greatest concern to food and feed safety. Mycotoxin contamination and crop diseases caused by Fusarium result from a combination of factors, including species of Fusarium, crop species/cultivar, other microbes, and the environment. We will use multiple approaches to identify critical components of Fusarium biology that contribute to crop diseases and mycotoxin contamination, with an emphasis on fumonisins produced by Fusarium verticillioides. We will use genomics to identify genetic markers that provide an unprecedented ability to identify diverse Fusarium species and to resolve phylogenetic relationships among species. We will also use genomics to elucidate the genetic potential of diverse Fusarium species to produce mycotoxins as well as the genetic mechanisms that affect distribution of mycotoxin biosynthetic genes. In addition, we will use mutagenesis to identify genes that suppress fumonisin production in F. verticillioides. Interactions of Fusarium and crops that lead to mycotoxin contamination likely result, in part, from metabolites produced by each organism. Thus, we will use mass spectrometry-based metabolomics to identify metabolites formed during the interaction of F. verticillioides and maize to determine which metabolites are critical for fumonisin contamination. We will also employ a transcriptomics approach to elucidate the effects of one class of plant metabolites, oxylipins, on fumonisin production in F. verticillioides. Because Fusarium mycotoxin levels are typically higher in crops with high levels of Fusarium-incited diseases, improving crop disease resistance will likely reduce mycotoxin contamination as well. Plant chitinases are enzymes that degrade chitin, an essential component of fungal cell walls, and likely contribute to fungal disease resistance. To elucidate how chitinases can be manipulated to improve this resistance, we will use proteomics to study the interaction of maize chitinases and fungal proteases that inactivate chitinases. We will also use classical mycological methods and DNA-based phylogenetic analyses to evaluate the range of fungal endophytes that occur in maize under diverse environmental conditions and to identify endophytes that can inhibit growth and/or fumonisin production in F. verticillioides. We will also use transcriptomics to determine the mechanism by which the fungal endophyte Talaromyces inhibits F. verticillioides. Finally, we will use quantitative polymerase chain reaction (PCR) to determine whether mycotoxin contamination contributes to the ability of F. verticillioides to compete with other maize-associated fungi.

Progress Report
Objective 1. Species of the fungus Fusarium are among the most agriculturally important fungi because they can cause crop diseases and they produce toxins (mycotoxins) that pose health hazards to humans, livestock and pets. We have used genome sequence analysis and other DNA sequence-based approaches to characterize the genetic diversity of two groups of Fusarium species called the Fusarium sambucinum species complex and the Fusarium tricinctum species complex. These complexes are among the most agriculturally important groups of Fusarium because of their collective abilities to cause destructive diseases and mycotoxin contamination in crops, particularly the cereals corn, barley, rice, sorghum and wheat. Based on the results of the genetic diversity studies, isolates of each species were selected and tested for their ability to produce mycotoxins and induce disease on wheat. Methods to identify pathogens in these two species complexes was significantly improved with the discovery that together they comprise 100 species, which is twice as many as previously determined. As part of the research, we have identified gene sequences that can be used to distinguish between Fusarium species. We have submitted the sequences to Fusarium MLST, an online database that is used by scientists worldwide to accurately identify species of Fusarium isolated from crops, wild plants, humans, and other animals. Genes responsible for biosynthesis of a mycotoxin can be used to assess the genetic potential of fungi to produce the mycotoxin, which in turn can contribute to assessments of the risks that the fungi pose to food and feed safety. We continued to determine the mycotoxin production potential in 187 Fusarium species using 344 genome sequences. We also continued analysis of how the genetic processes of horizontal gene transfer (i.e., direct transfer of genes from one species to another) and gene deletion have contributed to the distribution of mycotoxin biosynthetic genes among Fusarium species. We have found evidence that all Fusarium mycotoxin biosynthetic genes have been horizontally transferred at least once from one Fusarium species to another. Therefore, horizontal gene transfer has contributed to mycotoxin contamination problems caused by Fusarium species. As part of an effort to make USDA ARS data publicly available, we submitted over 100 Fusarium genome sequences to the GenBank database at the National Center for Biotechnology Information, which is part of the National Institutes of Health. These submissions have made USDA ARS the single biggest contributor of Fusarium genome sequences to the GenBank database. Government, university, and private-sector researchers can now access the sequences in GenBank and use them in diverse analyses of Fusarium biology, including analyses aimed at controlling crop diseases and mycotoxin contamination problems. The corn ear rot fungus Fusarium verticillioides is the predominant cause of fumonisin contamination in U.S. corn. The fungus has a poorly characterized genetic system that suppresses fumonisin production under most environmental conditions. Identification of the genes responsible for this suppression and understanding how they function should aid development of novel strategies that block fumonisin production in the fungus and thereby reduce contamination in corn. We have developed a genetically engineered strain of F. verticillioides that will facilitate identification of the fumonisin suppression genes and plan to make additional mutations. We will then use molecular genetic analyses to identify the mutated fumonisin suppression genes. Objective 2. The ability of F. verticillioides to contaminate corn with fumonisins and other mycotoxins is determined by the interaction of metabolites produced by both the fungus and the plant. Characterization of levels of these metabolites has potential to provide information and the identity of metabolites that can be used to develop mycotoxin control strategies for corn. The analysis of the thousands of metabolites produced by a single organism or multiple interacting organisms is called metabolomics. This year, we continued to develop metabolomic methods to monitor the thousands of metabolites that are produced during the interaction of corn and F. verticillioides. We used the metabolomic methods to analyze the metabolites produced by the two organisms under field conditions. We assessed metabolites produced in multiple corn tissues, including developing and mature kernels, infected with F. verticillioides. We have also improved our ability to identity individual metabolites (i.e., determine chemical structure) from among the thousands produced using commercially available metabolomics software and online databases of plant and fungal metabolites. We have also expanded our metabolomic analyses to other crop-Fusarium combinations that result in disease and toxin accumulation. Most of these latter efforts have focused on soybean and Fusarium virguliforme, the principle cause of soybean sudden death syndrome in the U.S. Objective 3. Fungal diseases of crops also result from specific interactions between fungal and plant proteins, especially extracellular proteins. Identifying these proteins and understanding their interactions can aid development of control strategies to reduce disease and mycotoxins in crops. During interactions between corn and ear rot fungi, corn produces an enzyme, ChitA, that degrades chitin in fungal cell walls, while the fungi produce enzymes, polyglycine hydrolases, that cleave and inactivate ChitA. We used mutational analysis to identify regions within the amino acid sequence of a polyglycine hydrolase that are required for the fungal enzyme’s ability to interact with and cleave ChitA. Some of the regions are crucial for enzyme stability, and others facilitate the physical interaction with ChitA. We also used our protein expression system in the yeast Pichia pastoris, to produce two polyglycine hydrolase from fungi that are not corn pathogens: the plant pathogen Fusarium solani and the wood rot fungus Galerina marginata. Both polyglycine hydrolases cleaved corn ChitA, but they cleaved different parts of ChitA than polyglycine hydrolase from corn pathogens. The functions of enzymes and other proteins are determined by both their amino acid sequence and how individual atoms within and among the amino acids interact to form a three-dimensional (3D) protein structure. Knowledge of an enzyme’s 3D structure provides valuable information about how it functions. Therefore, in collaboration with scientists at the University of Waterloo, Ontario, we continued to determine the 3D structure of polyglycine hydrolases using the technology known as X-ray crystallography. Because the technology requires crystal forms of proteins, we crystalized two polyglycine hydrolases. We used an approach that incorporates the chemical element selenium into the crystals, thereby enhancing the ability of X-ray crystallography to determine 3D structure. As a result, we are closer to determining the 3D structure of the polyglycine hydrolases. Analysis of corn genome sequences indicate that corn has multiple genes that encode ChitA-like proteins. Using our yeast protein expression system, we expressed and purified two of these proteins, ChitC and ChitD. We demonstrated that both proteins are cleaved by fungal polyglycine hydrolases, and we are in the process of determining the positions within ChitC and ChitD that are cleaved by the fungal enzymes. Objective 4. Little is known about how interactions among mycotoxin-producing fungi affect mycotoxin contamination of crops. However, such knowledge has potential to contribute to control practices that reduce mycotoxin contamination. To fill this knowledge gap, we are using a DNA-based system to assess competition, and its effect on mycotoxin accumulation, among three Fusarium species that cause corn ear rot: F. verticillioides and F. proliferatum, which produce fumonisins, and F. subglutinans, which produces the mycotoxins beauvericin and moniliformin. Thus far, we have focused on methods development; e.g., isolation of high-quality Fusarium DNA from infected seedlings and assessing spread of single species in the seedlings. Some strains of F. verticillioides have a variant (or allele) of a chromosomal region known as Spore Killer. During the sexual phase of the fungus’ lifecycle, spores that do not inherit the Spore Killer allele die. Thus, in genetic crosses of a strain with the Spore Killer allele and a strain that lacks the allele, only spores with the allele survive. We are developing a genetic system that will use the Spore Killer allele to spread a gene that blocks fumonisin production among individuals in field populations of F. verticillioides. If the fumonisin-blocking gene spreads sufficiently, most F. verticillioides strains in a field would not be able to produce the toxin, which in turn would reduce the levels of fumonisins in corn. As a prerequisite for developing the genetic system, we used targeted gene inactivation to demonstrate that a gene (SKC1) within the Spore Killer region is at least partially responsible for the spore-killing phenomenon. We are currently determining whether other genes or DNA sequences within the region also contribute to spore killing.

Accomplishments
1. Identification of fungal toxin genes facilitates risk assessment in food and feed. Sphinganine analog metabolites (SAMs) are a class of fungal toxins that have similar chemical structures and cause human and animal diseases by disrupting the metabolism of sphingolipids, a family of lipids essential for normal cellular functions. Except for fumonisins, which are among the mycotoxins of most concern to food and feed safety, nothing is known about the biochemical and genetic processes required for formation of SAMs. This lack of knowledge has prevented a thorough understanding of SAMs, including assessments of the health risks they pose and their role in the ecology of fungi. Therefore, ARS scientists in Peoria, Illinois, developed a method to identify SAM biosynthetic genes (i.e., genes responsible for production of fungal SAMs) using previously acquired knowledge on fumonisin biosynthetic genes. The method facilitated identification of biosynthetic genes for five structurally distinct groups of SAMs and demonstrated that the genetic potential to produce SAMs occurs widely but sporadically in fungi. Knowledge of SAM biosynthetic genes will aid assessments of risks that SAM-producing fungi pose to food and feed safety. The general approach used to identify SAM biosynthetic genes could be expanded to identify biosynthetic genes for other classes of fungal toxins, which would facilitate a wider assessment of health risks posed by toxin-producing fungi. Furthermore, identification of toxin biosynthetic genes facilitates determination of the role of the toxins in the ecology of fungi, which in turn can aid development of control strategies that reduce fungus-incited crop diseases and the associated mycotoxin contamination problems.

2. Development of diagnostic assay to assess potential of mycotoxin-producing fungi to overcome control practices. The fungus Fusarium includes some of the mycotoxin-producing and crop-disease-causing fungi of greatest concern to food and feed safety. Many species can undergo sexual reproduction, which can enhance their ability to adapt to changing environmental conditions, including their ability to overcome control practices aimed at reducing diseases and mycotoxin contamination in crops. In Fusarium, sexual reproduction (or mating) is controlled by two alternate forms of the chromosomal region known as the mating type locus; for two strains to mate, they must have different forms of the mating type locus. By analyzing the DNA sequences of the mating type locus in over 50 Fusarium species, ARS scientists in Peoria, Illinois, developed a robust DNA-based diagnostic assay to accurately determine the reproductive potential of isolates of the Fusarium fujikuroi species complex, one of the most agriculturally important groups of species within Fusarium. Because sexual reproduction can enhance the ability of fungi to overcome control practices, knowledge of the sexual reproductive potential of Fusarium species is important for development of robust control strategies that reduce disease and mycotoxins in crops. This in turn will reduce crop losses experienced by farmers and improve the safety of food and feed for the general public.

3. Identification of a genetic mechanism that controls production of trichothecene mycotoxins. Mycotoxins are fungal toxins that accumulate in crop plants and pose a health hazard to humans, pets, and livestock. Fungi have finely tuned genetic systems that control when and where they produce mycotoxins. In general, the same system controls production of a given mycotoxin among species in the same genus. However, ARS scientists in Peoria, Illinois, discovered a gene that controls trichothecene mycotoxin production in some but not all species of Fusarium. Analysis of diverse trichothecene-producing Fusarium species indicate that one group of species lost the gene while another group retained the gene. These findings indicate that genetic control of trichothecene production in Fusarium has undergone fundamental changes over time as groups of species have diverged from one another. The findings are significant, because one strategy to reduce mycotoxin contamination in crops is to block production of the toxins in fungi. Development of this strategy requires detailed knowledge of the genetic systems that control mycotoxin production in fungi. Thus, the findings have potential to contribute to control strategies that reduce trichothecene contamination in crops, thereby improving food and feed safety.

4. Discovery of a genetic system to protect against fumonisin mycotoxins in corn. Fumonisins are a group of fungal toxins (mycotoxins) that frequently contaminate corn, and as a result pose health risks to humans, pets, and livestock that eat corn-base food or feed. Because, fumonisins are toxic to a wide range of organisms, including fungi, fumonisin-producing fungi must have a mechanism to protect themselves from the mycotoxins. In collaboration with the Hans Knöll Institute in Germany, ARS scientists in Peoria, Illinois, identified a gene in the fumonisin-producing fungus Fusarium that protects the fungus from the toxic effects of fumonisins. The gene serves as the blueprint for an enzyme that is a resistant form of the enzyme that fumonisins normally inhibit to cause their toxic effects. These findings provide fundamental knowledge on how fungi protect themselves from the toxins they produce and has potential to contribute to strategies that protect humans and animals from the health hazards posed by fumonisins in corn.

5. A common enzymatic mechanism that leads to mycotoxin contamination in corn and a skin disease in humans. The bacterium Streptococcus pyogenes causes numerous skin and throat diseases. The bacterium produces an enzyme, ScpA, that cleaves a human immunity protein, thereby facilitating its ability to cause skin diseases. ARS researchers in Peoria, Illinois, discovered that the corn ear rot fungus Stenocarpella maydis produces an enzyme that is similar in protein sequence to ScpA and that cleaves the corn immunity protein ChitA. This finding indicates a shared enzymatic mechanism used by a bacterium to cause a human skin disease and a fungus to cause a corn disease that results in accumulation of mycotoxins. This knowledge contributes to the understanding of the processes that lead to crop diseases and in turn to mycotoxin contamination. Such knowledge will assist efforts of corn breeders to improve crop disease resistance and control mycotoxin contamination.

Review Publications
Naumann, T.A., Naldrett, M.J., Price, N.P.J. 2020. Kilbournase, a protease-associated domain subtilase secreted by the fungal corn pathogen Stenocarpella maydis. Fungal Genetics and Biology. 141:103399. https://doi.org/10.1016/j.fgb.2020.103399.
Brown, D.W., Villani, A., Susca, A., Moretti,A., Hao, G., Kim, H.-S., Proctor, R.H., McCormick, S.P., 2019. Gain and loss of a transcription factor that regulates late trichothecene biosynthetic pathway genes in Fusarium. Fungal Genetics and Biology. 136:103317. https://doi.org/10.1016/j.fgb.2019.103317.
Montoya-Martinez, A.C., Rodriguez-Alvarado, G., Fernandez-Pavia, S.P., Proctor, R.H., Kim, H.-S., O'Donnell, K. 2019. Design and validation of a robust multiplex polymerase chain reaction assay for MAT idiomorph within the Fusarium fujikuroi species complex. Mycologia. 111(5):772-781. https://doi.org/10.1080/00275514.2019.1649956.
Volpicella, M., Leoni, C., Fanizza, I., Distaso, M., Leoni, G., Farioli, L., Naumann, T., Pastorello, E., Ceci, L.R. 2017. Characterization of maize chitinase-A, a tough allergenic molecule. Allergy. 72(9):1423-1429. https://doi.org/10.1111/all.13164.
Laraba, I., Kim, H.-S., Proctor, R.H., Busman, M., O'Donnell, K., Felker, F.C., Aime, M.C., Koch, R.A., Wurdack, K.J. 2019. Fusarium xyrophilum, sp. nov., a member of the Fusarium fujikuroi species complex recovered from pseudoflowers on yellow-eyed grass (Xyris spp.) from Guyana. Mycologia. 112(1):39-51. https://doi.org/10.1080/00275514.2019.1668991.
Wipfler, R., McCormick, S.P., Proctor, R., Teresi, J., Hao, G., Ward, T., Alexander, N., Vaughan, M.M. 2019. Synergistic phytotoxic effects of culmorin and trichothecene mycotoxins. Toxins. 11(10):555. https://doi.org/10.3390/toxins11100555.
Vaughan, M.M., Ward, T.J., McCormick, S.P., Orwig, N., Hay, W.T., Proctor, R., Palmquist, D. 2020. Intrapopulation antagonism can reduce the growth and aggressiveness of the wheat head blight pathogen Fusarium graminearum. Phytopathology. 110(4):916-926. https://doi.org/10.1094/PHYTO-09-19-0341-R.
Cardoza, R.E., McCormick, S.P., Lindo, L., Kim, H.-S., Olivera, E.R., Nelson, D.R., Proctor, R.H., Gutierrez, S. 2019. A cytochrome P450 monooxygenase gene required for biosynthesis of the trichothecene toxin harzianum A in Trichoderma. Applied Microbiology and Biotechnology. 103(19):8087-8103. https://doi.org/10.1007/s00253-019-10047-2.
Proctor, R.H., McCormick, S.P., Gutierrez, S. 2020. Genetic bases for variation in structure and biological activity of trichothecene toxins produced by diverse fungi. Applied Microbiology and Biotechnology. 104:5185–5199. https://doi.org/10.1007/s00253-020-10612-0.
Janevska, S., Luliia, F., Rautschek, J., Hoefgen, S., Proctor, R.H., Hillman, F., Valiante, V. 2020. Self-protection against the sphingolipid biosynthesis inhibitor fumonisin B1 is conferred by a FUM cluster-encoded ceramide synthase. mBio. 11(3):e00455-20. https://doi.org/10.1128/mBio.00455-20.
Kim, H.S., Lohmar, J.M., Busman, M., Brown, D.W., Naumann, T.A., Divon, H.H., Lysoe, E., Uhlig, S., Proctor, R.H. 2020. Identification and distribution of gene clusters required for synthesis of sphingolipid metabolism inhibitors in diverse species of the filamentous fungus Fusarium. Biomed Central (BMC) Genomics. 21:510. https://doi.org/10.1186/s12864-020-06896-1.