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

Location: Mycotoxin Prevention and Applied Microbiology Research

2017 Annual Report

1a. 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.

1b. 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.

3. Progress Report:
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.

4. Accomplishments
1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

Review 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.