Location: Mycotoxin Prevention and Applied Microbiology Research
2021 Annual Report
Objectives
Objective 1: Use comparative phylogenomic approaches to enable accurate identification of mycotoxigenic Fusarium and to elucidate components of Fusarium genomes that are responsible for variation in mycotoxin production. Sub-objectives 1.1 through 1.3 are as follows: 1.1 – Develop a DNA sequence database that facilitates accurate identification of all toxigenic Fusarium species; 1.2 – Determine whether mycotoxin biosynthetic gene clusters and genetic networks that regulate cluster expression differ in their distributions among Fusarium species; 1.3 – Determine whether F. verticillioides has genes that repress fumonisin production.
Objective 2: Develop and utilize liquid chromatography-mass spectrometry (LC-MS) approaches for metabolomic analysis of Fusarium verticillioides infection of maize.
Sub-objective 2.1 and 2.2 are as follows: 2.1 – Develop workflows for untargeted analyses of the metabolomes of maize, F. verticillioides, and the maize-F. verticillioides interaction; and 2.2 – Identify metabolic biomarkers for high and low levels of F. verticillioides-induced disease in maize.
Objective 3: Identify and characterize plant and fungal factors that can impact mycotoxin contamination via their effects on plant disease development. Sub-Objective 3.1 through 3.4 are as follows: 3.1 – Determine how primary sequence and secondary structure of fungal polyglycine hydrolases affect the inhibitory activity of this class of proteases against plant chitinases; 3.2 – Isolate and identify ChitA alloform-specific proteases secreted by the fungi Stenocarpella maydis and Trichoderma viride; 3.3 – Elucidate the role of plant class IV chitinases in maize-fungus interactions; and 3.4 – Identify candidate receptor and regulatory genes that mediate oxylipin-induced changes in expression of fumonisin biosynthetic genes and fumonisin production in F. verticillioides.
Objective 4: Identify and characterize components of fungus-fungus interactions that contribute to or inhibit mycotoxin contamination of crops. Sub-objective 4.1 through 4.3 are as follows:
4.1 – Sample across different climate zones to identify novel fungal endophytes of maize that inhibit growth and/or fumonisin production in F. verticillioides; 4.2 – Identify candidate genes in Talaromyces that are responsible for inhibition of growth in F. verticillioides; and 4.3 – Determine whether production of fumonisins and other mycotoxins contributes to the competitiveness of F. verticillioides with other Fusarium species.
Approach
The fungus Fusarium is of concern to agriculture because it can cause crop diseases and produce mycotoxins, including three (fumonisins, trichothecenes, and zearalenone) that are among the mycotoxins of greatest concern to food and feed safety. Mycotoxin contamination and crop diseases caused by Fusarium result from a combination of factors, including species of Fusarium, crop species/cultivar, other microbes, and the environment. We will use multiple approaches to identify critical components of Fusarium biology that contribute to crop diseases and mycotoxin contamination, with an emphasis on fumonisins produced by Fusarium verticillioides. We will use genomics to identify genetic markers that provide an unprecedented ability to identify diverse Fusarium species and to resolve phylogenetic relationships among species. We will also use genomics to elucidate the genetic potential of diverse Fusarium species to produce mycotoxins as well as the genetic mechanisms that affect distribution of mycotoxin biosynthetic genes. In addition, we will use mutagenesis to identify genes that suppress fumonisin production in F. verticillioides. Interactions of Fusarium and crops that lead to mycotoxin contamination likely result, in part, from metabolites produced by each organism. Thus, we will use mass spectrometry-based metabolomics to identify metabolites formed during the interaction of F. verticillioides and maize to determine which metabolites are critical for fumonisin contamination. We will also employ a transcriptomics approach to elucidate the effects of one class of plant metabolites, oxylipins, on fumonisin production in F. verticillioides. Because Fusarium mycotoxin levels are typically higher in crops with high levels of Fusarium-incited diseases, improving crop disease resistance will likely reduce mycotoxin contamination as well. Plant chitinases are enzymes that degrade chitin, an essential component of fungal cell walls, and likely contribute to fungal disease resistance. To elucidate how chitinases can be manipulated to improve this resistance, we will use proteomics to study the interaction of maize chitinases and fungal proteases that inactivate chitinases. We will also use classical mycological methods and DNA-based phylogenetic analyses to evaluate the range of fungal endophytes that occur in maize under diverse environmental conditions and to identify endophytes that can inhibit growth and/or fumonisin production in F. verticillioides. We will also use transcriptomics to determine the mechanism by which the fungal endophyte Talaromyces inhibits F. verticillioides. Finally, we will use quantitative polymerase chain reaction (PCR) to determine whether mycotoxin contamination contributes to the ability of F. verticillioides to compete with other maize-associated fungi.
Progress Report
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.
Accomplishments
1. Determination of 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.
2. 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.
3. 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.
4. 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.
5. 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.
Review 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.
