Progress 01/19/16 to 01/04/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. The research project focused on Fusarium, a fungus of major agricultural concern because of the collective abilities of its many species to cause destructive diseases of diverse economically important plants and to contaminate food and feed with toxins. The presence of Fusarium toxins in food and feed crops pose a health hazard to humans, pets, and livestock. The project included four objectives that focused on assessing the threat that Fusarium species pose to food/feed safety and biosecurity and on identifying biological or chemical factors that can be used to develop strategies to reduce toxin contamination in crops. Fumonisins are among the toxins of most concern to food and feed safety because of their toxicity combined with their frequent and widespread occurrence in corn. Therefore, several of the project⿿s objectives and/or subobjectives focused on identifying factors with potential to reduce fumonisin contamination in corn. Objective 1 of the project focused on two aspects of Fusarium: 1) development of methods for accurate identification of toxin-producing Fusarium species; and 2) identification of components of Fusarium genomes that are responsible for variation in toxin production. To address this objective, we generated genome sequences for over 250 fungal strains that represent the known range of genetic diversity that exists in Fusarium. The resulting sequences were used to determine how the strains were related to one another and to identify DNA sequences that can be used to determine species identity of unknown isolates of Fusarium. The sequences were also used to predict toxin production abilities of strains. This was possible because genes required for formation of most Fusarium toxins (i. e., toxin biosynthetic genes) have been identified. Therefore, toxin production potential of each strain was determined by assessing which toxin biosynthetic genes were present in the genome sequence of each strain. In many cases, toxin production by the strains was confirmed using standard chemical analyses. One major accomplishment of this research was the submission of over 100 Fusarium genome sequences to the GenBank database. Their presence in GenBank makes the sequences web- accessible in support of efforts worldwide to monitor and control diseases and toxin contamination caused by diverse species of Fusarium. To address objective 1, we also developed a genetic method to identify genes that suppress fumonisin production in Fusarium verticillioides, a species that is the major cause of fumonisin contamination in corn in many parts of the world. Identification of these genes and elucidation of genetic mechanisms that block fumonisin production in F. verticillioides are needed to develop control strategies that prevent the fungus from contaminating corn with fumonisins. Objective 2 of the project focused on development of methods to detect and quantify the thousands of metabolites (chemical compounds) produced by F. verticillioides and corn during interactions of these two organisms that lead to fumonisin contamination. The rationale for this objective was that the ability of F. verticillioides to cause fumonisin contamination in corn is mediated, in part, by the interaction of metabolites produced by the two organisms. Knowing what the metabolites are and at what levels they are produced should provide insight into metabolites that contribute to or inhibit fumonisin contamination and that could serve as markers in breeding and other programs aimed at reducing fumonisin contamination in corn. Over the course of this project, we developed methods to monitor thousands of metabolites that are produced by F. verticillioides and corn when the organisms were grown separately or together. The methods were used to analyze metabolites produced in F. verticillioides-infected corn seedlings and developing kernels in the laboratory and in the field. We were able to identity many of the metabolites (i.e., determine chemical structure) from among the thousands produced. To do this we used commercially available software and online databases of plant and fungal metabolites. Near the end of the project, we expanded the analyses to include interactions of soybean and Fusarium virguliforme, the principle cause of soybean sudden death syndrome (SDS) in the U.S. Thus, the method can be adapted to find metabolic markers for other fungus-plant interactions that result in crop disease and/or toxin contamination. Objective 3 of the project focused on identifying corn and fungal factors that impact toxin contamination through their effects on crop disease development. Proteins are one group of these factors. Fungal diseases of plants are mediated by interactions between proteins produced by fungi and plants, especially extracellular proteins. Identifying these proteins and understanding their interactions are needed to develop control strategies to reduce disease and toxin contamination of crops. During this project, we determined how multiple fungal proteins, collectively known as polyglycine hydrolases, inactivate a corn protein (ChitA) that degrades the carbohydrate walls that surround and protect fungal cells. We identified how some amino acids that make up the polyglycine hydrolase Bz-cmp, produced by the corn pathogen Bipolaris zeicola, facilitate inactivation of ChitA. We searched for and found proteins similar to Bz- cmp in other fungi and showed that these other proteins had reduced activity against the corn ChitA, indicating that Bz-cmp is adapted to be highly effective at inactivating the corn ChitA. We also identified two additional corn proteins (ChitC and ChitD) that degrade fungal cell walls and showed that multiple fungal pathogens of corn produce polyglycine hydrolases that degrade ChitC and ChitD. These findings revealed the complexity of interactions between fungal and corn pathogens and indicate that there are multiple potential targets that can be exploited to enhance resistance of corn to fungal diseases and toxin contamination. Knowledge of the three-dimensional structure of proteins can provide valuable insights into their functions and how they interact with other proteins. 3D structure information can, for example, aid in understanding why two proteins differ in activity. To obtain additional insights into how fungal polyglycine hydrolases function, along with collaborators in Canada we initiated experiments to examine the 3D structure of two polyglycine hydrolases using a technology known as X-ray crystallography. This technology requires crystallized proteins, and formation of the crystals requires relatively large quantities of purified protein. As a result, much of the research at ARS was aimed at developing methods to produce and purify large quantities of the two polyglycine hydrolases so that they could be crystallized. These methods have proven successful and analysis of the 3D structures of the crystalized proteins is on-going in Canada. There is a growing body of knowledge to indicate that interactions between Fusarium species can reduce disease and toxin contamination. However, whether such interactions between Fusarium species affect fumonisin contamination is not known. To begin to address this question, Objective 4 of the project focused on competition between fumonisin- producing and nonproducing species of Fusarium in corn. As part of this research, we developed a DNA-based method to accurately measure the growth of three Fusarium species in corn. All three species are pathogens of corn but two, F. verticillioides and F. proliferatum, produce fumonisins, and the third, F. subglutinans, does not. Results of experiments demonstrated that when grown in pairwise combinations, F. proliferatum was more competitive than the other two species, but F. verticillioides was not more competitive than F. subglutinans. Experiments are in progress to determine how these different levels of competitiveness of the fungi affect fumonisin contamination. For information on additional progress during the current fiscal year, see the progress report for the report for project 5010-42000-053-00D, which replaces this recently expired project. Record of Any Impact of Maximized Teleworking Requirement: Scientific research has multiple phases, including experimental design, performing experiments, and data analysis. Because maximized teleworking requirements limited access to ARS laboratories, it allowed employees to devote more time to experimental design and data analysis, activities that can be done in a home office with the aid of a computer and access to scientific literature. As a result, scientists had more time to develop project plans and contribute to grant proposals. In addition, technical staff were encouraged to participate in experimental design and data analysis, which increased the diversity of perspectives contributing to the design and analysis. In contrast, maximized teleworking requirements severely inhibited scientists and technical staff from performing experiments in laboratories. The reduced occupancy requirements limited the number of experiments that could be done, and the experiments could be done only by employees willing and authorized to work on-site. ACCOMPLISHMENTS 01 Determination what fungi cause toxin contamination in wheat. The fungus Fusarium is a global agricultural concern because it causes destructive crop diseases and contaminates crops with toxins that are health hazards to humans, pets, and livestock. A group of species known as the Fusarium sambucinum species complex (FSAMSC) is of particular concern because it includes species that cause the wheat disease Fusarium head blight (FHB) and that produce trichothecenes, one of the toxin classes of greatest concern to food and feed safety worldwide. ARS researchers at Peoria, Illinois, investigated a global collection of 171 fungal strains selected to represent the known range of genetic diversity of FSAMSC. The results indicate the strains comprised 74 genetically distinct species that can be categorized into six groups based on the types of toxins they produce. The results also indicate that species in only two of the groups can cause FHB and trichothecene contamination of wheat. These findings help clarify which Fusarium species cause disease and toxin contamination of wheat. Such information is critical for development of effect strategies to control these agricultural problems. 02 Unraveling details of how fungal proteins disarm corn defense proteins. The ability of fungi to cause disease and toxin contamination in corn is mediated by proteins produced by both organisms. ARS researchers at Peoria, Illinois, used fine-tuned mutation analyses to unravel details of how a protein (Bz-cmp) produced by the fungus Bipolaris zeicola inactivates a corn protein that degrades fungal cell walls. The analyzes identified some of the individual amino acids within Bz-cmp that physically interact with the corn protein to inactivate it. The researchers also discovered that two fungi that are not corn pathogens produce proteins that are similar to Bz-cmp but exhibit only low levels of activity against the corn protein. These findings expand understanding of the molecular interactions that occur between fungi and crops and determine whether fungi can cause disease and contamination of crops with toxins. Knowledge of variability in Bz-cmp proteins produced by fungi will contribute to identification of naturally occurring and/or engineering of corn proteins that are not affected by Bz-cmp proteins. This in turn will enhance resistance of corn to fungal diseases and toxin contamination. 03 Elucidating genetic bases for variation in toxin production in fungi. The presence of fungal toxins in food and feed crops pose a health threat to humans, pets, and livestock. But the threat posed by some fungi is not uniform because of variation in toxin production among strains within individual species. Researchers at ARS, Peoria, Illinois, in collaboration with the National Research Council, Italy, and Cape Peninsula University of Technology, South Africa, investigated the genetic basis for variation in toxin production abilities in two fungal species. In Fusarium verticillioides strains that produce only low levels of fumonisin toxins, all genes necessary for toxin formation are intact, but expression of the genes is low compared to strains that produce high levels of fumonisins. In Aspergillus westerdijkiae, strains that do not produce ochratoxins lack one of the genes necessary for toxin formation. The findings for A. westerdijkiae are being used to design a DNA-based assay to rapidly monitor the presence of ochratoxin-producing and nonproducing strains of Aspergillus under field conditions. Further, the nonproducing strains of A. westerdijkiae are being evaluated to determine whether they can be used in biological control strategies to reduce ochratoxin contamination in food. 04 Discovery of novel toxin producing fungi. Although many of the fungi that cause disease and toxin contamination problems in crops are known, the causes of some problems have yet to be determined. ARS researchers at Peoria, Illinois, and collaborators in Argentina, Mexico, and Tunisia characterized strains of the fungus Fusarium isolated from economically important grasses and trees, including mango. DNA sequence- based identification methods revealed the grass isolates comprised two novel species of Fusarium while the tree isolates were a previously characterized species. One species from grasses produced T-2 toxin, which is of major concern worldwide and occurred at high levels in pasture grass from which the species was isolated. These findings expand knowledge of the diversity of Fusarium species with potential to cause disease and/or toxin contamination of food and feed crops. The knowledge will aid efforts of plant disease specialists and quarantine officials who are focused on preventing the introduction of novel toxin- producing fungi into the U.S. 05 Defining Fusarium, one of the world⿿s most agriculturally destructive fungi. The fungal genus Fusarium consists of hundreds of genetically distinct species that collectively threaten agriculture by causing economically important diseases of most crops and by producing toxins that pose health risks to humans, pets, and livestock. Further, some species can also cause human infections under certain conditions. Despite these food/feed safety, agricultural biosecurity, and medical problems, the range of species that are considered to be Fusarium has not been uniformly accepted by research and regulatory institutions tasked with monitoring and solving the problems these toxin-producing fungi cause. Therefore, ARS researchers at Peoria, Illinois, and researchers at 115 institutions worldwide conducted a large-scale DNA sequence analysis that clarified what fungal species make up the genus Fusarium. The Results provide a scientifically sound basis for including diverse species within Fusarium in a manner that is consistent with historical precedence. The results will also significantly foster exchange of accurate information about Fusarium among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and researchers. This exchange will enhance efforts to develop strategies to reduce crop diseases, toxin contamination, and medical problems caused by Fusarium.
Impacts
(N/A)
Publications
- Beccaccioli, M., Salustri, M., Scala, V., Ludovici, M., Cacciotti, A., D'Angeli, S., Brown, D.W., Reverberi, M. 2021. The effect of Fusarium verticillioides fumonisins on fatty acids, sphingolipids, and oxylipins in maize germlings. International Journal of Molecular Sciences. 22(5). Article 2435. https://doi.org/10.3390/ijms22052435.
- Dowd, P.F., Naumann, T.A., Johnson, E.T., Price, N.P. 2020. A maizewin protein confers enhanced antiinsect and antifungal resistance when the gene is transgenically expressed in maize callus. Plant Gene. 24. Article 100259. https://doi.org/10.1016/j.plgene.2020.100259.
- Lilly, M., Rheeder, J.P., Proctor, R.H., Gelderblom, W.C.A. 2021. FUM gene expression and variation in fumonisin production of clonal isolates of Fusarium verticillioides MRC 826. World Mycotoxin Journal. 14(2):121-137. https://doi.org/10.3920/WMJ2020.2626.
- Laraba, I., McCormick, S.P., Vaughan, M.M., Geiser, D.M., O'Donnell, K. 2021. Correction: Phylogenetic diversity, trichothecene potential, and pathogenicity within Fusarium sambucinum species complex. PLoS ONE. 16(4). Article e0250812. https://doi.org/10.1371/journal.pone.0250812.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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. 03 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. 04 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. 05 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.
Impacts
(N/A)
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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Objective 1: Species of Fusarium are among the most agriculturally important fungi because they can cause crop diseases and they produce toxins (mycotoxins) that are harmful to the health of humans and livestock. The Fusarium sambucinum species complex (FSAMSC) is a group of closely related species and includes the most economically important producers of trichothecene mycotoxins. Despite this, many species within FSAMSC have not been characterized, and their economic impact and trichothecene production abilities have not been determined. To fill this knowledge gap, we characterized a collection of 173 isolates from around the world that, based on preliminary evidence, were likely members of FSAMSC. Analysis of DNA sequence data for multiple genes from the isolates indicated that FSAMSC comprises over 70 genetically distinct species, which is twice as many species as documented previously. Most of the isolates in the collection produced trichothecenes, and 10 of the isolates that were determined to be novel species caused high levels of wheat head blight, one of the most important wheat diseases worldwide. Given their ability to produce trichothecene mycotoxins and to cause head blight, some of the novel species have potential to affect wheat production. Genes responsible for biosynthesis of a mycotoxin can be used to assess the genetic potential of fungi to produce the mycotoxin. In collaboration with researchers at the University of Minnesota, we assessed the genetic potential of over 700 species of diverse fungi to produce fumonisin mycotoxins by examining the occurrence of fumonisin biosynthetic genes in publicly available genome sequence databases and an ARS database. Our results indicate that the occurrence of fumonisin biosynthetic genes is rare among fungi; the genes are present in some species of Fusarium, the saprophytic fungus Aspergillus, the insect pathogen Tolypocladium, the corn leaf blight pathogen Bipolaris, and the tomato pathogen Alternaria. We also confirmed fumonisin production by chemical analysis in some of the Alternaria, Aspergillus, Fusarium and Tolypocladium strains. The findings indicate that despite the common occurrence of fumonisins in crops particularly corn, production of fumonisins is rare among fungi. This in turn indicates that efforts to control fumonisin contamination in corn should continue to focus on contamination caused by Fusarium. The corn ear rot fungus Fusarium verticillioides is the predominant cause of fumonisin contamination, which is a worldwide concern. The finding that F. verticillioides does not produce fumonisins under some conditions indicates the fungus has a genetic system that suppresses fumonisin production. Identification of the genes that cause this suppression and understanding how they function could lead to novel strategies that reduce fumonisin contamination of corn. We developed a mutation approach to identify genes in F. verticillioides that are responsible for suppression of fumonisin production. The approach is designed to identify mutant strains of the fungus in which the suppression genes have been inactivated, and as a result the mutants should produce fumonisins under conditions that normally suppress production. Using the approach, we have identified 300 potential mutant strains of F. verticillioides, and we are currently examining the mutants by chemical analyses to determine whether fumonisin production is no longer suppressed. Multiple genes regulate (turn on) mycotoxin production in fungi. Using a database consisting of genome sequences, we examined the occurrence of a subset of these regulatory genes in over 150 Fusarium species. We found that the genes were present in all species examined, however some species had multiple copies of some genes. The widespread occurrence of regulatory genes that affect mycotoxin production indicates that mycotoxin control efforts that target regulatory genes have the potential to be effective for multiple fungi and mycotoxins. Fumonisins belong to a class of compounds called sphinganine analog metabolites (SAMs) that inhibit synthesis of sphingolipids, which are an essential component of cell membranes in plants and animals. Because sphingolipids are essential, inhibiting their synthesis causes multiple toxic effects that in turn lead to diseases such as those induced by fumonisins. The inhibition of sphingolipid biosynthesis results from similarities in chemical structures of SAMs and sphinganine, a metabolic intermediate in sphingolipid biosynthesis. We identified five sets of SAM biosynthetic genes in Fusarium. Each set is likely responsible for synthesis of a structurally distinct SAM. We also determined that one or more sets of SAM biosynthetic genes occur in 35% of Fusarium species. However, the occurrence of the genes varied among different lineages (i.e. , groups of closely related species) of Fusarium. Knowledge of the chemical structures of the SAMS and their roles in the ecology of Fusarium species that produce them should aid in development of strategies to reduce fumonisin contamination in crops. Objective 2. The ability of the fungus Fusarium to cause mycotoxin contamination in crop plants is most likely determined by the interaction of metabolites produced by both the fungus and plants. Identification of these metabolites and understanding their interactions has potential to contribute to development of strategies that reduce mycotoxin contamination and crop diseases caused by Fusarium. To this end, we developed an analytical chemistry method to detect and identify the thousands of metabolites that are produced during the interaction of corn and Fusarium verticillioides. The analysis of the thousands of metabolites produced by a single organism or multiple interacting organisms is called metabolomics. Our metabolomics method is based on two technologies: liquid chromatography, which separates metabolites based on their chemical properties; and mass spectrometry, which detects metabolites based on their mass and how they decompose. This year, we developed metabolomic protocols for analysis of developing corn kernels, mature kernels, and other corn tissues infected with F. verticillioides. We have also improved our ability to identify metabolites using commercially available metabolomics software and on-line databases of plant and fungal metabolites. We have also expanded our metabolomic analyses to examine the interaction of soybean and Fusarium virguliforme, the principle cause of soybean sudden death syndrome in the U.S. Objective 3. The ability of fungi to cause crop diseases and mycotoxin contamination is affected by interactions of enzymes produced by plants and fungi. Understanding the functions of these enzymes should contribute to development of strategies to control crop diseases and mycotoxin contamination. Chitin is an essential component of fungal cell walls and plants produce chitin-degrading enzymes (chitinases) that inhibit fungal growth. However, some fungi produce chitinase degrading enzymes to protect themselves from plant chitinases. This year, we continued to characterize a chitinase-degrading enzyme produced by the fungus Epicoccum sorghi. We focused on producing purified enzyme to facilitate determination of its three-dimensional molecular structure using x-ray technology. Such structural information can be used to determine which parts of an enzyme bind to its substrate and/or have catalytic activity. We genetically engineered a yeast strain to produce high levels of the E. sorghi enzyme. The increased production facilitated incorporation of selenium atoms into the enzyme, which will aid in molecular structure determination. We also continued to characterize a chitinase-degrading activity produced by the corn ear rot pathogen Stenocarpella maydis. We purified an enzyme with the chitinase-degrading activity from cultures of S. maydis. This facilitated determination of the amino acid sequence of part of the enzyme using mass spectrometry technology. We compared the resulting data to genome sequence data of S. maydis to identify two proteins that are likely responsible for the chitinase-degrading activity. We are currently developing a system to produce high levels of purified enzyme in yeast and a bacterium. Objective 4. Mycotoxin contamination in crops can be affected by microbial communities that occur on the crops. As a result, microorganisms in these communities have potential to aid in control of mycotoxin contamination. Fungi that colonize plants without causing disease (i.e., endophytic fungi) are part of plant-associated microbial communities. The diversity of endophytic fungi in corn and their ability to suppress fumonisin contamination in this crop are poorly understood. To address this knowledge gap, we isolated fungi from corn kernels produced in three regions that represent three different climate zones in which corn is grown in the US (Minnesota, Kansas and Texas). Characterization of fungal isolated from Texas identified 10 distinct species of the fungus Talaromyces. However, none of the Talaromyces isolates examined inhibited growth or fumonisin production in F. verticillioides. Fungi that cause plant disease and produce mycotoxins are also part of plant-associated microbial communities. However, little is known about how interactions between these fungi affect mycotoxin contamination. Such knowledge has potential to inform efforts to reduce mycotoxin contamination. To fill this knowledge gap, we developed a DNA-based system to assess competition among three Fusarium species that cause corn ear rot: F. verticillioides and F. proliferatum and F. subglutinans. In pairwise combinations, F. proliferatum grows more rapidly than either F. subglutinans or F. verticillioides on autoclaved corn kernels. We are currently assessing the ability of these fungi to compete with one another in corn. Accomplishments 01 Update of critical information on reference strains of the toxin- producing fungus Fusarium. Species of the fungus Fusarium are a food/ feed security concern because they cause plant diseases, and they are a food/feed safety concern because they produce multiple toxins (mycotoxins) that are health hazards to humans and animals. Despite these concerns, there is considerable confusion with respect to which species of Fusarium produce which mycotoxins. ARS researchers in Peoria, Illinois, used state-of-the-art technologies to determine species identities (DNA-sequence-based identification) and mycotoxin production abilities (mass spectrometry-based chemical analyses) of a collection of 158 Fusarium strains described in a 1984 compendium used as a reference by extension agents, diagnosticians and researchers worldwide who deal with crop diseases and mycotoxin contamination problem caused by Fusarium. The results indicated that half of the strains were misidentified, and that some mycotoxins were produced by genetically diverse species, whereas others were produced by only one or a few groups of closely related species. These findings provide clarity to Fusarium-related food safety concerns because they accurately connect species identity and mycotoxin production for a collection of diverse species that have served as an important reference for mycotoxin production in Fusarium for 35 years. 02 Identification of components of enzymatic warfare between plants and fungi. Physical, chemical, and enzymatic interactions between plants and pathogenic fungi can result in a disease when a fungus overcomes plant defenses or no disease when a plant blocks fungal infection. For example, corn produces enzymes (chitinases) that inhibit fungal growth by degrading an essential component of fungal cell walls known as chitin. ARS researchers in Peoria, Illinois, identified an enzyme (protease) produced by the fungus Stenocarpella maydis that degrades a corn chitinase and determined how the protease cleaves the chitinase. The results contribute to a worldwide effort to elucidate critical chemical and enzyme components of plant-fungus interactions that can be used to develop strategies to control crop disease and mycotoxin contamination problems. 03 Determination of genetic processes that cause variation in toxin production among fungal species. Species of the fungus Fusarium negatively impact agriculture by causing crop diseases and by producing toxins (mycotoxins) that are health hazards to humans, pets, and livestock. Using a genome sequence approach, ARS scientists in Peoria, Illinois, determined the distribution of genes responsible for production of mycotoxins and other biologically active compounds (e.g., pigments) in 35 Fusarium species. A series of analyses that measure variation in DNA sequences also revealed evidence that the current distribution of the genes has resulted from three genetic processes: 1) inheritance of genes from parent to offspring over many generations; 2) loss of genes by deletion or mutation; and 3) direct transfer of genes from one species to another in a manner analogous to transfer of antibiotic resistance genes among bacterial species. The results provide evidence for the genetic mechanisms that are responsible for differences in mycotoxin production among Fusarium species as well as the frequency with which the mechanisms occur. In addition, the genes can be used as genetic markers to assess the occurrence mycotoxin- producing fungi in crops, and they are potential targets for control efforts aimed at reducing mycotoxin contamination and/or diseases caused by Fusarium.
Impacts
(N/A)
Publications
- Lindo, L., McCormick, S.P., Cardoza, R.E., Kim, H.-S., Brown, D.W., Alexander, N.J., Proctor, R.H., Gutierrez, S. 2018. Role of Trichoderma arundinaceum tri10 in regulation of terpene biosynthetic genes and in control of metabolic flux. Applied and Environmental Microbiology. 122:31- 46.
- Vrabka, J., Niehaus, E.-M., Munsterkotter, M., Proctor, R.H., Brown, D.W., Novak, O., Pencik, A., Tarkowska, D., Hromadova, K., Hradilova, M. 2019. Production and role of hormones during interaction of Fusarium species with maize (Zea mays L.) seedlings. Frontiers in Plant Science.
- Naumann, T.A., Price, N.P.J. 2018. Purification and in vitro activities of a chitinase-modifying protein from the corn ear rot pathogen Stenocarpella maydis. Physiological and Molecular Plant Pathology 106:74-80.
- Lindo, L., McCormick, S.P., Cardoza, R.E., Busman, M., Alexander, N.J., Proctor, R.H., Gutierrez, S. 2018. Requirement of two acyltransferases for 4-O-acylation during biosynthesis of Harzianum A, an antifungal trichothecene produced by Trichoderma arundinaceum. Journal of Agricultural and Food Chemistry. 67(2):723-734.
- Munkvold, G.P., Weieneth, L., Proctor, R.H., Busman, M., Blandino, M., Susca, A., Logrieco, A., Moretti, A. 2018. Pathogenicity of fumonisin- producing and nonproducing strains of Aspergillus species in section Nigri to maize ears and seedlings. Plant Disease. 102(2):282-291.
- Jacobs-Venter, A., Laraba, I., Geiser, D.M., Busman, M., Vaughan, M.M., Proctor, R.H., McCormick, S.P., O'Donnell, K. 2018. Molecular systematics of two sister clades, the Fusarium concolor and F. babinda species complexes, and the discovery of a novel microcycle macroconidium⿿producing species from South Africa. Mycologia. 110:1189-1204.
- O'Donnell, K., McCormick, S.P., Busman, M., Proctor, R.H., Ward, T.J., Doehring, G., Geiser, D.M., Alberts, J.F., Rheeder, J.P. 2018. Marasas et al. 1984 "toxigenic Fusarium species: identity and mycotoxicology" revisited. Mycologia. 110(6):1058-1080.
- Villani, A., Proctor, R.H., Kim, H.-S., Brown, D.W., Logrieco, A.F., Amatulli, M.T., Moretti, A., Susca, A. 2019. Variation in secondary metabolite production potential in the Fusarium incarnatum-equiseti species complex revealed by comparative analysis of 13 genomes. BMC Genomics. 20:314.
- Pereira, C.B., Ward, T.J., Tessmann, D.J., Del Ponte, E.M., Laraba, I., Vaughan, M.M., McCormick, S.P., Busman, M., Kelly, A., Proctor, R.H., O'Donnell, K. 2018. Fusarium subtropicale sp. nov., a novel nivalenol mycotoxin-producing species isolated from barley (Hordeum vulgare) in Brazil and sister to F. praegraminearum. Mycologia. 110(5):860-871.
- Kim, W., Cavinder, B., Proctor, R.H., O'Donnell, K., Townsend, J.P., Trail, F. 2019. Comparative genomics and transcriptomics during sexual development gives insight into the life history of the cosmopolitan fungus Fusarium neocosmosporiellum. Frontiers in Microbiology.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. The following describes research that addresses Objective 1. To address knowledge gaps in the phylogenetic diversity and relationships of Fusarium, and in their ability to produce mycotoxins, whole genome sequencing was used to produce a database consisting of over 300 genome sequences that represent approximately 250 species of Fusarium. Researchers are using the database to address critical questions related to Fusarium mycotoxins, pigments, and other metabolites, which are collectively referred to as secondary metabolites. Most genes responsible for synthesis of the same secondary metabolite are located adjacent to one another along a chromosome; i.e., in a gene cluster. Using the Fusarium genome sequence database, researchers are determining which Fusarium species have previously described gene clusters responsible for synthesis of mycotoxins and other secondary metabolites. This analysis revealed marked differences in distributions of the clusters. Researchers are also using phylogenetic analyses of gene clusters to elucidate the genetic and evolutionary processes that contribute to differences in cluster distribution. Researchers also used the genome sequences to identify five Fusarium gene clusters that are most likely responsible for synthesis of metabolites with chemical structures similar to those of fumonisin mycotoxins. Collaborative research with scientists in Norway demonstrated that one of the clusters is responsible for synthesis of a fumonisin-like metabolite known as 2-Amino-14,16-dimethyloctadecan-3-ol. This research demonstrates that knowledge about known metabolite gene clusters can contribute to identification of metabolic products of other gene clusters. Researchers retrieved genes from 51 fusaria represented in the Fusarium genome sequence database to elucidate the genetic diversity and evolutionary relationships of species within Fusarium fujikuroi species complex (FFSC) and several closely related species complexes. A phylogenetic analyses of 16 genes using a maximum likelihood approach was conducted. The analyses revealed that fumonisin-producing fusaria included in the study were nested within three major clades that correspond to the previously described African, American, and Asian clades of FFSC. The DNA sequence data generated in this study provide a new and valuable resource for developing diagnostic tools for detection and identification of fumonisin-producing and nonproducing species of Fusarium. A collection of 158 Fusarium strains that have served as references for Fusarium researchers worldwide for over four decades were re-evaluated. Researchers used a modern DNA sequence-based phylogenetic approach to clarify the species identity of the strains, and state-of-the-art mass spectrometry-based analytical methods to determine the mycotoxin production ability of the strains. The strains were originally reported in �Toxigenic Fusarium Species,� a book that was published in 1984. The DNA-based analysis revealed that species diversity of the collection is far greater than previously recognized. The analytical chemistry analyses clarified the ability of the strains to produce nine classes of mycotoxins, including trichothecenes and fumonisins. Clarification of the identity and mycotoxin production ability should improve the utility of the strains to the Fusarium research community. The DNA sequence data generated for this study were incorporated into Fusarium MLST, an online database that is used by researchers worldwide to identify Fusarium. The following describes research that addresses Objective 2. The ability of Fusarium to cause mycotoxin contamination and crop diseases is likely determined by the interaction of metabolites produced by the fungus and its host plants. Identification of these metabolites and understanding their interactions has the potential to contribute to development of strategies that reduce mycotoxin contamination and crop diseases caused by Fusarium. Thus, researchers are developing analytical methods to monitor the thousands of metabolites produced during the interaction of the fumonisin-producing species Fusarium verticillioides and corn, its principal host. The wholesale analysis of thousands of metabolites produced by a single organism or multiple interacting organisms is called metabolomics. Researchers are developing metabolomic methods that combine liquid chromatography (LC) and tandem mass spectrometry (MS) to detect and distinguish between metabolites based on differences in molecular weight and fragmentation patterns in the mass spectrometer. This year researchers developed protocols for metabolomic analysis of uninfected maize seedlings and maize seedlings infected with F. verticillioides and to improve our ability to identify metabolites using commercially available metabolomics software. In addition, researchers extended metabolomic studies to examine the interaction of soybean seedlings and Fusarium virguliforme, the principal cause of soybean sudden death syndrome in the U.S. The following describes research that addresses Objective 3. The ability of fungi to cause mycotoxin contamination and diseases of crops is affected by interactions of enzymes produced by these organisms. Knowledge of the interactions has potential to contribute to development of strategies to control mycotoxin contamination and crop diseases. Some plants produce enzymes (chitinases) that degrade chitin, an essential component of fungal cell walls. Some fungi can counteract plant chitinases by producing chitinase modifying proteins (CMPs) that inactivate chitinases by cleaving them into two or more pieces. The corn chitinase ChitA is thought to provide a successful defense against some fungi. However, it is not clear whether the effectiveness of ChitA occurs by direct inhibition of fungal growth, or by releasing chitin fragments that elicit a plant defense response that in turn inhibits fungi. This year, in order to test these two competing hypotheses, researchers developed methods to produce milligram quantities of pure ChitA and the two peptides resulting from the cleavage of ChitA by the CPM produced by Stenocarpella maydis, a fungus that is a major cause of corn ear rot worldwide. Mutated forms of ChitA were also produced in which its amino acid sequence was changed to reduce its enzymatic activity. The purified functional ChitA, its CPM cleavage products, and mutated forms of ChitA are being used to assess their ability to directly inhibit fungal growth and their ability to elicit a plant defense response in corn. Researchers have also successfully produced milligram quantities of the S. maydis CPM. The purified CPM is being used in experiments to determine the biochemical mechanism by which it cleaves ChitA. This research will provide insight into how plants successfully defend themselves during some plant-fungus interactions and how some fungi overcome plant defenses during other interactions. Such insights will contribute to plant breeding efforts aimed at enhancing resistance to fungal-incited diseases and the mycotoxin contamination problems associated with the diseases. The following describes research that addresses Objective 4. Mycotoxin contamination and diseases of crops are affected by microbial communities that occur in the crops. Therefore, there is potential for microorganisms in these communities to be used to control crop diseases and mycotoxin contamination. One component of the microbial communities are endophytic fungi; i.e., fungi that colonize plant tissue without causing disease symptoms. The diversity of endophytic fungi in corn and their ability to suppress mycotoxin contamination and disease in this crop are poorly understood. Therefore, we are examining the diversity of endophytic fungi in corn grown in different climate zones in the U.S and examining their ability to inhibit fumonisin production in F. verticillioides. This year, researchers analyzed corn from a northern and a central climate zone, and found marked differences in the species composition of fungal endophytes in the two zones. One of the endophytic fungi from corn was identified as Talaromyces stollii and shown to inhibit fumonisin production in F. verticillioides. A strain of T. stollii is being subjected to genome sequencing and transcriptomic analyses to elucidate the mechanism by which it inhibits fumonisin production. Accomplishments 01 Zeroing in on fungi responsible for fumonisin mycotoxin contamination in corn. Fumonisins are among the mycotoxins of greatest concern to food and feed safety because they are frequent contaminants in corn, have potential to cause esophageal cancer in adults and neural tube defects in newborns, and can cause multiple diseases in some domestic animals. Although the fungus Fusarium verticillioides has been considered the primary cause of fumonisin contamination in corn for decades, the recent finding that another corn-associated fungus, Aspergillus niger, can produce fumonisins has raised concerns that it too is responsible for fumonisin contamination. In a multiyear collaboration with scientists at Iowa State University, ARS scientists in Peoria, Illinois, demonstrated that infection of corn ears with A. niger did not result in accumulation of significant levels of fumonisins. This finding indicates that A. niger does not contribute significantly to fumonisin contamination in corn. Therefore, the finding also indicates that government, university, and private-sector scientists working to prevent fumonisin contamination in this important crop should focus their efforts on F. verticillioides. 02 A method for reducing production of fumonisin mycotoxins. Fumonisins are among the mycotoxins of greatest concern to food safety because of their frequent occurrence as contaminants in corn combined with their potential to cause esophageal cancer in adults and neural tube defects in newborns. The fumonisin-producing fungus Fusarium verticillioides is frequently recovered from corn and is the primary cause of fumonisin contamination in this crop. ARS scientists in Peoria, Illinois, demonstrated that a natural cell defense system known as RNAi (ribonucleic acid interference) can be harnessed to block fumonisin production in cultures of F. verticillioides by suppressing activation of genes in the fungus that are required for synthesis of fumonisins. The finding demonstrates the potential of RNAi technology as a control strategy to reduce contamination of crops with fumonisins and other mycotoxins. 03 Development of methods for measuring Fusarium mycotoxins and other metabolites. Species of the fungus Fusarium pose a dual threat to agriculture because they can cause economically important diseases of most crop plants, and they can produce mycotoxins that are harmful to the health of humans and domestic animals. In addition to mycotoxins, Fusarium species produce other biologically active metabolites such as pigments and plant hormones. Methods to detect and accurately measure these other metabolites are essential in understanding whether they contribute to the ability of Fusarium to cause crop diseases and/or mycotoxin contamination. In a series of studies, an ARS scientist in Peoria, Illinois, developed methods to detect and measure 12 classes of Fusarium metabolites. The methods are based on a combination of state- of-the-art technologies known as liquid chromatography and mass spectrometry. These methods provide government, university, and private- sector researchers tools to investigate whether and how the metabolites contribute to plant disease and or mycotoxin contamination. The methods will also aid researchers to assess the levels of the metabolites in crops and to determine whether the metabolites pose health risks to humans and animals. 04 Discovery of a novel toxin-producing pathogen of wheat. Species of the fungus Fusarium pose a serious threat to food production worldwide because they cause economically devastating crop diseases that reduce crop yield and quality. Many Fusarium species can also contaminate crops with mycotoxins that pose health risks to humans and domestic animals. In collaboration with an Algerian scientist from the Higher National Agronomic School in Algiers, ARS scientists in Peoria, Illinois, discovered a novel species of Fusarium that was recovered from diseased stems of wheat plants in Algeria. The scientists formally described the novel species as Fusarium algeriense, and demonstrated that it could cause a wheat stem disease (crown rot), and that it could produce two types of mycotoxins. These findings will be of use to plant quarantine officials and plant disease specialists charged with preventing introduction of foreign pathogens into the U.S. The results will also inform wheat breeders and pathologists that a greater diversity of Fusarium species can cause crown rot of wheat than was previously recognized. This in turn has the potential to affect wheat breeding and other efforts aimed at reducing crown rot.
Impacts
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Publications
- Peterson, S.W., Jurjevic, Z. 2018. New species of Talaromyces isolated from maize, indoor air, and other substrates. Mycologia. 109(4):537-556.
- Weber, J.M., Ryan, K.N., Tandon, R., Mathur, M., Girma, T., Steiner-Asiedu, M., Saalia, F., Zaidi, S., Soofi, S., Okos, M., Vosti, S.A., Manary, M.J. 2017. Acceptability of locally produced ready-to-use therapeutic foods in Ethiopia, Ghana, Pakistan and India. Maternal and Child Nutrition. 13(2). doi:10.1111/mcn.12250.
- Laraba, I., Keddad, A., Boureghda, H., Abdallah, N., Vaughan, M.M., Proctor, R.H., Busman, M., O'Donnell, K. 2018. Fusarium algeriense, sp. nov., a novel toxigenic crown rot pathogen of durum wheat from Algeria is nested within the Fusarium burgessii species complex. Mycologia. 106(6) :935-950.
- Waalwijk, C., Taga, M., Zheng, S.-L., Proctor, R.H., Vaughan, M.M., O'Donnell, K. 2018. Karyotype evolution in Fusarium. IMA Fungus. 9(1):13- 26. doi:10.5598/imafungus.2018.09.01.02
- Price, N.P.J., Hartman, T.M., Li, J., Velpula, K.K., Naumann, T.A., Guda, M.R.,Yu, B., Bischoff, K.M. 2017. Modified tunicamycins with reduced eukaryotic toxicity that enhance the antibacterial activity of �-lactams. Journal of Antibiotics. 70(11):1070-1077. doi: 10.1038/ja.2017.101.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Fusarium is among the fungi of greatest concern to agricultural production and food/feed safety because it causes crop diseases and can contaminate infected crops with toxins (mycotoxins) that are harmful to the health of humans and livestock animals. Because the fungus is estimated to consist of hundreds of species, including many that are difficult to distinguish by traditional identification methods, tools are needed for reliable and rapid identification of species as well as for discerning relationships among species and groups of species. There are also critical gaps in knowledge with respect to which species produce which mycotoxins, plant growth regulators, and other metabolites that impact agricultural production. To address these knowledge gaps, ARS scientists in Peoria, Illinois, are generating and analyzing the complete genome sequences (i.e., DNA sequence of all chromosomes and extra- chromosomal elements) of a large collection of Fusarium isolates. The sequences are being used in two major ways: 1) to identify genetic markers that can more accurately identify species; and 2) to determine what mycotoxins and other metabolites each species has the potential to produce. To date, the scientists have generated genome sequences for over 250 Fusarium species. The information is being combined in order to assess the frequency with which Fusarium species exchange genes responsible for mycotoxin synthesis and with which the genes degenerate. This information provides insight into how mycotoxin production abilities of a species can change and, therefore, how the risks that species pose to food safety change. This research addresses Objective 1 of the project. Over the past half century, chemists and biologists have produced a large body of literature on mycotoxin production in thousands of isolates of the fungus Fusarium. In most of this literature, Fusarium isolates were identified based on morphological traits. However, recent DNA sequence-based analyses demonstrate that morphology-based analyses of Fusarium frequently misidentify species and underestimate species diversity within this genus. As a result, there is significant confusion in the literature as to which Fusarium species produce which mycotoxins. To clarify some of the confusion, ARS scientists in Peoria, Illinois, are analyzing 145 isolates of Fusarium from the South African Medical Research Council Culture Collection (MRC) that formed the basis of a book published in 1984 that is one of the most cited references on mycotoxin production in Fusarium. The ARS scientists are conducting DNA-based phylogenetic analyses to evaluate species identity and diversity of the 145 isolates. The scientists are also analyzing the ability of the isolates to produce a diversity of mycotoxins using state-of-the-art analytical chemistry methods that were not used in the original 1984 publication. In addition, the ARS scientists are generating and analyzing genome sequences of several novel species that were identified among the 145 isolates. The results of these analyses will provide much needed clarity on the diversity and identity of mycotoxin-producing species of Fusarium. This research addresses Objective 1 of the project. A critical factor in determining whether crops become contaminated with mycotoxins is the regulation of mycotoxin production in fungi: that is, the genetic and biochemical processes that induce or suppress fungal genes responsible for synthesis of mycotoxins. Understanding factors that regulate mycotoxin synthesis has tremendous potential for development of control strategies to prevent mycotoxin contamination of crops. Therefore, ARS scientists in Peoria, Illinois, are conducting two lines of research to dissect regulation of mycotoxin synthesis in Fusarium. The goal of one line is to identify genes in Fusarium verticillioides that suppress synthesis of fumonisin mycotoxins. To this end, the ARS scientists are developing laboratory strains of the fungus in which genetic suppression of fumonisin synthesis has been interrupted. Once strain development is complete, the genes responsible for suppression can be identified and characterized to provide new insights into how fungi control mycotoxin production within their cells. The goal of the other line of research is to determine how a novel gene in Fusarium controls synthesis of trichothecene mycotoxins. The latter research has demonstrated that the gene induces expression of some but not all trichothecene biosynthetic genes. This result combined with the finding that the gene is present in some trichothecene-producing species but not in others indicates there has been a recent and fundamental change in the genetic regulation of trichothecene production in Fusarium. This research addresses Objective 1 of the project. The ability of the fungus Fusarium to cause mycotoxin contamination and crop diseases is likely determined by the interaction of chemical compounds (metabolites) produced by both the fungus and its host plants. Identification of these metabolites and understanding their interactions should contribute to development of strategies to control both mycotoxin contamination and crop diseases caused by Fusarium. To identify the metabolites, ARS scientists in Peoria, Illinois, are developing analytical methods to monitor the extraordinarily broad range of metabolites produced by Fusarium and its hosts. Such wholesale analysis of the hundreds or even thousands of metabolites is called metabolomics. The methods developed in this research are based on two technologies: 1) liquid chromatography (LC), which separates metabolites from one another; and 2) mass spectrometry (MS), which detects metabolites based on their weight. This year, methods were developed for processing of samples prior to analysis, for detecting metabolites produced by Fusarium grown in laboratory cultures, and for statistical analysis of the large datasets resulting from each experiment. These methods have been used in two ways: 1) untargeted analyses to generate information on the broad range of metabolites produced by the species Fusarium verticillioides; and 2) targeted analyses to determine what mycotoxins are produced by newly recognized species of Fusarium. One aspect of the targeted analyses was development of methods for measurement of Fusarium mycotoxins in crops. As part of this, a method was developed to measure levels of the Fusarium metabolite 8-O-methylbostrycoidin in corn. This research addresses Objective 2 of the project. The ability of fungi to cause crop diseases and mycotoxin contamination is affected by the interaction of enzymes produced by the fungi and crop plants. Understanding such interactions should contribute to development of strategies that reduce crop diseases and mycotoxin contamination. One of the enzyme interactions is centered on chitin, which is an essential carbohydrate component of fungal cell walls. Plants produce chitin- degrading enzymes (chitinases), while in order to protect themselves fungi produce chitinase modifying proteins (CMPs) that inactivate chitinases. Previously, ARS scientists in Peoria, Illinois, determined that developing ears of corn produce multiple forms of the chitinase ChitA, and that the corn ear rot fungus Stenocarpella maydis produces a CMP that inactivates some but not all forms of ChitA. However, the S. maydis protein responsible for the CMP activity was not identified. This year, therefore, the scientists continued research aimed at identifying the S. maydis CMP protein as well as the gene that encodes the protein. To this end, they developed methods to produce CMP in cultures of the fungus, to extract the CMP protein from the cultures, and to measure CMP activity. They have also generated a genome sequence for S. maydis, which will facilitate identification of the CMP gene once the protein is identified and characterized. This research addresses objective 3 of the project. The ability of fungi to cause mycotoxin contamination and disease in crops can be affected by the presence of other microorganisms, including fungi that colonize plants without causing disease (i.e., endophytic fungi). In some cases, endophytic fungi can suppress disease and/or mycotoxin accumulation. As a result, endophytic fungi have potential for development of control strategies to reduce mycotoxin contamination and diseases of crops. Relatively little is known about the diversity of endophytic fungi in corn or their potential to suppress mycotoxin contamination and diseases caused by the fungus Fusarium. Therefore, ARS scientists in Peoria, Illinois, are examining the diversity of endophytic fungi in corn cultivated in different climate zones of the continental U. S. This year�s investigations revealed that two genera of endophytic fungi, Talaromyces and Sarocladium, dominate in corn grown in climate zone 1. At least 15 species of Talaromyces were recovered, including three previously undescribed species. Further analysis revealed that some of the Talaromyces isolates inhibit production of fumonisin mycotoxins by the fungus Fusarium verticillioides. This research addresses objective 4 of the project. The ecological advantage provided by most mycotoxins to the fungi that produce them is not known. However, understanding the advantages should contribute to development of control strategies to reduce mycotoxin contamination in crops. One possibility is that mycotoxins allow fungi to compete with other microorganisms. Therefore, ARS scientists in Peoria, Illinois, are conducting research to determine whether production of fumonisin mycotoxins by the corn ear rot fungus Fusarium verticillioides enhances its competitiveness with other species of Fusarium that also occur in corn. This year, the ARS scientists have developed DNA-based methods to rapidly distinguish and measure the biomass of F. verticillioides and two other Fusarium species. Accomplishments 01 Global genetic diversity of a fungus that causes Fusarium crown rot and Fusarium head blight of wheat. The fungus Fusarium culmorum causes Fusarium crown rot (FCR) and Fusarium head blight (FHB), two of the most important wheat diseases worldwide. In addition, FHB usually results in contamination of wheat with trichothecene mycotoxins, which pose health risks to humans and livestock animals. Although F. culmorum occurs widely in wheat growing regions of the world, little is known about its genetic diversity. Therefore, in collaboration with scientists at The National School of Agronomy (El Harrach, Algeria), The Institute of Sciences of Food Production (Bari, Italy), the Grains Research and Development Corporation (Canberra, Australia), and Pennsylvania State University, ARS scientists in Peoria, Illinois, carried out research to assess the genetic diversity of isolates of F. culmorum recovered from the US, Algeria, Australia and Italy. The analyses identified two widely occurring subgroups (populations) of the fungus that overlap in their geographic distribution. Both populations consisted of individuals that produce one of two types of trichothecenes: 3-acetyl-deoxynivalenol (3ADON) or nivalenol (NIV). In addition, both populations had genes that enable Fusarium to undergo sexual reproduction and, thereby, increase its genetic diversity. The improved understanding of the genetic diversity of F. culmorum will aid plant breeders in development of wheat cultivars with broad-based resistance to FCR and FHB. Icreased FHB resistance should reduce the levels of trichothecene mycotoxins in wheat, which in turn will improve the yield, quality, and safety of wheat. 02 A novel Fusarium species that produces mycotoxins and causes head blight of wheat. Multiple species of Fusarium cause Fusarium head blight (FHB), one of the most economically important diseases of wheat worldwide. Species that cause FHB can also typically contaminate infected grain with trichothecene mycotoxins that pose health risks to humans and livestock animals. In collaboration with scientists at the Grain Research Laboratory (Winnipeg, Canada) and Landcare Research (Auckland, New Zealand), ARS scientists in Peoria, Illinois, characterized a novel species of Fusarium and formally named it Fusarium praegraminearum. The scientists demonstrated that the fungus can cause FHB and that it can produce two types of trichothecene mycotoxins (4-acetylnivalenol and 4,15-diacetylnivalenol), as well as zearalenone, a mycotoxin that inhibits reproduction in swine. DNA sequence-based phylogenetic analyses revealed that F. praegraminearum occupies a unique position in the phylogenetic diversity of FHB-causing Fusarium species. The results of this research have been used to update DNA-based diagnostic tests to include information on F. praegraminearum. The results will be of interest to plant quarantine officials and plant disease specialists charged with preventing introduction of foreign pathogens into the United States. The results can also be used by plant breeders working to generate cereal crops with broad resistance to FHB and to reduce trichothecene contamination. 03 Genetic causes of variation in mycotoxin biosynthetic genes in fungi. Fungi vary in their ability to produce mycotoxins that pose health risks to humans and livestock animals. The causes of such variation are currently the subject of intense study because they have the potential to provide insight into how to prevent mycotoxin contamination in food and feed crops, as well as insight into genetic changes that alter the ability of fungi to cause mycotoxin contamination and crop diseases. To gain such insight, ARS scientists in Peoria, Illinois, collaborated with scientists at the Norwegian Institute of Bioeconomy (As, Norway) and at The Ohio State University to assess the distribution of genes responsible for synthesis of the mycotoxin depudecin in genome sequences of over 550 fungi, including 32 species of the fungus Fusarium. The analysis revealed that depudecin biosynthetic genes are present in highly diverse fungi, but that their occurrence varies markedly among species, even among closely related species. The analysis also revealed three main genetic causes for the observed distribution of depudecin genes: 1) passage of the genes from parents to offspring; 2) direct transfer of the genes from one species to another; and 3) degeneration of the genes. These findings provide fundamental insights into genetic mechanisms that affect the distribution of mycotoxin biosynthetic genes. The findings can be used for development of strategies to reduce mycotoxin contamination in crops and for evaluation of changes in risks that mycotoxin-producing fungi pose to human and animal health. 04 Genetic bases for variation in mycotoxin production within fungal species. Aspergillus niger and Aspergillus welwitschiae are fungal species that occur naturally on many food crops. Typically, they do not cause crop diseases; however, they can occasionally cause seed and/or fruit rot on some crops. Concerns about the safety of these fungi have arisen with the discovery that some strains of both species produce the mycotoxins fumonisins and ochratoxins. ARS scientists in Peoria, Illinois, in collaboration with scientists at the Institute of Sciences of Food Production (Bari, Italy), investigated the frequency of fumonisin and ochratoxin production in both species and assessed the genetic causes of variation in mycotoxin production within each species. The investigation revealed variation in the frequency of fumonisin- and ochratoxin-producing strains in both species. The investigation also revealed that the genetic cause of fumonisin nonproduction in A. welwitschiae and ochratoxin nonproduction in both species was loss of genes responsible for synthesis of the toxins. These results expand the understanding of the genetic causes of intra-species variation in mycotoxin production in fungi. In addition, the results will help guide academic, government, and private-sector research aimed at developing strategies to reduce mycotoxin contamination in crops. The results will also assist regulatory agencies that assess the risks that fungi pose to human and animal health. 05 Improving food safety by identifying a food allergen in corn. Allergens are proteins that trigger immune system reactions resulting in symptoms such as nausea, digestive problems, headaches, hives and/or swollen airways. Although corn is not among the most common food allergens, it does nevertheless cause allergic reactions that cause mild to severe symptoms in a small percentage of people. An important aspect of research on food allergens is the identification and characterization of proteins that induce allergic reactions. Therefore, in collaboration with scientists at the Institute of Biomembranes (Bari, Italy), ARS scientists in Peoria, Illinois, identified a corn kernel protein that is a food allergen. The allergen is an enzyme that degrades an essential component of fungal cell walls. The scientists also showed that this enzyme is stable at high temperatures and under acidic environments, characteristics that would enable it to remain allergenic after cooking and passage through the stomach. Identifying this protein as a food allergen will aid in the medical diagnosis of food allergies, will enhance understanding of why some food proteins trigger undesirable immune responses, and thereby improve food safety.
Impacts
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Publications
- Grafenhan, T., Johnston, P.R., Vaughan, M.M., McCormick, S.P., Proctor, R. H., Busman, M., Ward, T.J., O'Donnell, K. 2017. Fusarium praegraminearum sp. nov. is a novel nivalenol mycotoxin-producing pathogen from New Zealand can induce head blight on wheat. Mycologia. 108(6):1229-1239.
- Peterson, S.W. 2017. Targeting conserved genes in Penicillium species. In: Moretti, A., Susca, A., editors. Mycotoxigenic Fungi: Methods and Protocols. New York, NY: Humana Press. p. 149-157.
- Johnson, E.T., Skory, C.D., Naumann, T.A., Jairajpuri, M.A., Dowd, P.F. 2016. Three sorghum serpin recombinant proteins inhibit midgut trypsin activity and growth of corn earworm. Agri Gene. 2(2016):11-16. doi:10.1016/ j.aggene.2016.09.005.
- Naumann, T.A., Bakota, E.L., Price, N.P.J. 2017. Recognition of corn defense chitinases by fungal polyglycine hydrolases. Protein Science. 26(6) :1214-1223.
- Susca, A., Proctor, R.H., Morelli, M., Haidukowski, M., Gallo, A., Logrieco, A.F., Moretti, A. 2016. Variation in fumonisin and ochratoxin production associated with differences in biosynthetic gene content in Aspergillus niger and A. welwitschiae isolates from multiple crop and geographic origins. Frontiers in Microbiology. doi: 10.3389/fmicb.2016. 01412.
- Price, N.P.J., Labeda, D.P., Naumann, T.A., Vermillion, K.E., Bowman, M.J., Berhow, M.A., Metcalf, W.W., Bischoff, K.M. 2016. Quinovosamycins: New tunicamycin-type antibiotics in which the alpha, beta-1", 11'-linked N- acetylglucosamine residue is replaced by N-acetylquinovosamine. Journal of Antibiotics. 69(8):637-646. doi: 10.1038/ja.2016.49.
- Edwards, J., Auer, D., de Alwis, S.-K., Summerell, B., Aoki, T., Proctor, R.H., Busman, M., O'Donnell, K. 2017. Fusarium agapanthi sp. nov, a novel bikaverin and fusarubin-producing leaf and stem spot pathogen of Agapanthus praecox (African lily) from Australia and Italy. Mycologia. 108(5):981-992.
- Laraba, I., Boureghda, H., Abdallah, N., Bouaicha, O., Obanor, F., Moretti, A., Geiser, D.M., Kim, H.-S., McCormick, S.P., Proctor, R.H., Kelly, A.C., Ward, T.J., O'Donnell, K. 2017. Population genetic structure and mycotoxin potential of the wheat crown rot and head blight pathogen Fusarium culmorum in Algeria. Fungal Genetics and Biology. 103:34-41.
- Reynolds, H.T., Slot, J.C., Divon, H.H., Lysoe, E., Proctor, R.H., Brown, D.W. 2017. Differential retention of gene functions in a secondary metabolite cluster. Molecular Biology and Evolution. doi: 10.1093/molbev/ msx145.
- Proctor, R.H., Vaughan, M.M. 2017. Targeting fumonisin biosynthetic genes. In: Moretti, A., Susca, A., editors. Mycotoxigenic Fungi. Vol 1542. New York, NY: Springer. p. 201-214.
- Niehaus, E.-M., Munsterkotter, M., Proctor, R.H., Brown, D.W., Sharon, A., Idan, Y., Oren-Young, L., Sieber, C.M., Novak, O., Pencik, A., et al. 2016. Comparative �omics� of the Fusarium fujikuroi species complex highlights differences in genetic potential and metabolite synthesis. Genome Biology and Evolution. 8(11):3574�3599.
- Brown, D.W., Baker, S.E. 2017. Mycotoxins: A fungal genomics perspective. In: Moretti, A., Susca, A., editors. Mycotoxigenic Fungi. Vol 1542. New York, NY: Springer. p. 367-379.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Project 5010-42000-050-00D replaces Project 5010-42000-044-00D and has a start date of 01/19/2016. Scientists have begun planning and conducting research to fulfill the 12-month milestones. Fusarium is among the fungi of greatest concern to agricultural production and food/feed safety because it causes crop diseases and can contaminate infected crops with toxins (mycotoxins) that are harmful to humans and livestock. Because the fungus is estimated to consist of hundreds of species, including many that are difficult to distinguish morphologically, tools are needed for reliable and rapid identification of species as well as for discerning relationships among species and groups of species. There are also critical gaps in knowledge with respect to which species produce mycotoxins, plant growth regulators, and other metabolites of agricultural concern. ARS scientists in Peoria, Illinois, are taking a comparative genomics approach to identify the needed tools and to fill the knowledge gaps. That is, they are determining the complete DNA sequence of all chromosomes and extra-chromosomal elements in individual species of Fusarium, and then comparing the sequences from different species. To date, the scientists have generated such sequences (also known as genome sequences) for 170 Fusarium species, and are evaluating 35 genes that are present in all the sequences for their utility as genetic markers to distinguish between species and to determine how species are related to one another. The scientists are also using the genome sequences to determine the genetic potential of species to produce 24 chemically distinct families of mycotoxins, plant growth regulators, and other metabolites of agricultural concern. This research addresses Objective 1 of the project, which is to use comparative genomics approaches for accurate identification of mycotoxigenic Fusarium species and to elucidate components of Fusarium genomes that are responsible for variation in mycotoxin production. The fungi Aspergillus niger and Aspergillus welwitschiae are of concern to food safety because they are used for fermentation of food and beverages, they occur widely on food crops in the field and in storage, and they can produce the mycotoxins fumonisins and ochratoxins, which are hazardous to humans and livestock. In collaboration with scientists at the National Research Council, Italy, ARS scientists in Peoria, Illinois, have determined that even though A. niger and A. welwitschiae are considered to be ochratoxin-producing species, the majority of strains isolated from a wide range of crops do not produce the mycotoxin. The scientists also demonstrated that the five genes directly responsible for synthesis of ochratoxin are present in ochratoxin-producing strains of both Aspergillus species, but absent in ochratoxin-nonproducing strains. This research is related to Objective 1 of the project, a major focus of which is to determine components of genomes that are responsible for variation in mycotoxin production. The ability of Fusarium to cause crop disease and, thereby, mycotoxin contamination is likely determined by the interaction of metabolites produced by the fungus and its host plants. To identify these metabolites, ARS scientists in Peoria, Illinois, are developing an analytical method to monitor all metabolites produced by Fusarium verticillioides and its host plant, maize (corn), during development of ear rot disease and accumulation of fumonisin mycotoxins. This approach (also known as metabolomics) uses state-of-the-art chromatography and mass spectrometry technologies to separate and provide a highly accurate estimate of mass for each metabolite produced by an organism(s). This year, the scientists developed protocols for extraction of metabolites from pure cultures of F. verticillioides, chromatography-mass spectrometry analysis of the extracts, and analysis of the substantial amount of resulting data. The protocols have facilitated detection of approximately 10,000 metabolites produced by F. verticillioides, a remarkable and unprecedented number for this fungus. This research is the first step in development of metabolomic methods to examine the F. verticillioides-maize interaction. This research addresses Objective 2 of the project, the focus of which is to develop and utilize liquid chromatography-mass spectrometry (LC-MS) approaches for metabolomic analysis of F. verticillioides infection of maize. Chitin is an essential component of fungal cell walls, and plants produce chitin-degrading enzymes (chitinases) to resist fungal infection. Fungi, in turn, produce enzymes that degrade chitinases (i.e., chitin modifying proteases, or CMPs) to defend themselves against plant chitinases. To elucidate the mechanisms by which CMPs degrade chitinases, and therefore determine whether chitinases can be modified to resist CMPs, ARS scientists in Peoria, Illinois, are determining which amino acid residues in CMPs are required for activity. Using a CMP produced by the corn pathogen Epicoccum sorghi as a model system as well as a mutagenesis approach, the scientists are determining how replacement of specific amino acids within the E. sorghi CPM affects the chitinase-degrading activity of the enzyme. The results indicate that replacement of the amino acid tryptophan with the amino acid alanine at several positions in the CMP reduces activity by affecting three dimensional structure of the enzyme and, thereby, its ability to interact with chitinase. This research addresses Objective 3 of the project, a major focus of which is to characterize fungal CPMs. The ability of Fusarium and other fungi to cause disease and mycotoxin contamination in crop plants can be affected by the presence of other fungi in the plants, including fungi that colonize plants without causing disease (i.e., endophytic fungi). In some cases, endophytic fungi can suppress disease and/or mycotoxin accumulation caused by plant pathogenic fungi. Relatively little is known about the diversity of endophytic fungi in maize (corn) or their potential to suppress disease and mycotoxin contamination caused by Fusarium. The endophytic fungus Sarocladium zeae occurs in maize, and some strains inhibit F. verticillioides, a prominent cause of ear rot and fumonisin mycotoxin contamination in maize. To determine whether genetic differences within S. zeae are associated with the ability to inhibit F. verticillioides, ARS scientists in Peoria, Illinois, are examining variation in DNA sequences of six genes among 50 isolates of S. zeae obtained from Illinois maize or culture collections. To date, results of the analysis indicate that maize isolates of S. zeae can be grouped into 4 genetically distinct groups, and that members of at least one group inhibit F. verticillioides. This research addresses Objective 4 of the project, a major focus of which is to identify fungal endophytes of maize and assess their ability to inhibit F. verticillioides.
Impacts
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Publications
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