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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

2018 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:
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.

4. Accomplishments
1. 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.

2. 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.

3. 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.

4. 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.

Review 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 ß-lactams. Journal of Antibiotics. 70(11):1070-1077. doi: 10.1038/ja.2017.101.