Location: Mycotoxin Prevention and Applied Microbiology Research
2019 Annual Report
Objectives
Objective 1: Use comparative phylogenomic approaches to enable accurate identification of mycotoxigenic Fusarium and to elucidate components of Fusarium genomes that are responsible for variation in mycotoxin production. Sub-objectives 1.1 through 1.3 are as follows: 1.1 – Develop a DNA sequence database that facilitates accurate identification of all toxigenic Fusarium species; 1.2 – Determine whether mycotoxin biosynthetic gene clusters and genetic networks that regulate cluster expression differ in their distributions among Fusarium species; 1.3 – Determine whether F. verticillioides has genes that repress fumonisin production.
Objective 2: Develop and utilize liquid chromatography-mass spectrometry (LC-MS) approaches for metabolomic analysis of Fusarium verticillioides infection of maize.
Sub-objective 2.1 and 2.2 are as follows: 2.1 – Develop workflows for untargeted analyses of the metabolomes of maize, F. verticillioides, and the maize-F. verticillioides interaction; and 2.2 – Identify metabolic biomarkers for high and low levels of F. verticillioides-induced disease in maize.
Objective 3: Identify and characterize plant and fungal factors that can impact mycotoxin contamination via their effects on plant disease development. Sub-Objective 3.1 through 3.4 are as follows: 3.1 – Determine how primary sequence and secondary structure of fungal polyglycine hydrolases affect the inhibitory activity of this class of proteases against plant chitinases; 3.2 – Isolate and identify ChitA alloform-specific proteases secreted by the fungi Stenocarpella maydis and Trichoderma viride; 3.3 – Elucidate the role of plant class IV chitinases in maize-fungus interactions; and 3.4 – Identify candidate receptor and regulatory genes that mediate oxylipin-induced changes in expression of fumonisin biosynthetic genes and fumonisin production in F. verticillioides.
Objective 4: Identify and characterize components of fungus-fungus interactions that contribute to or inhibit mycotoxin contamination of crops. Sub-objective 4.1 through 4.3 are as follows:
4.1 – Sample across different climate zones to identify novel fungal endophytes of maize that inhibit growth and/or fumonisin production in F. verticillioides; 4.2 – Identify candidate genes in Talaromyces that are responsible for inhibition of growth in F. verticillioides; and 4.3 – Determine whether production of fumonisins and other mycotoxins contributes to the competitiveness of F. verticillioides with other Fusarium species.
Approach
The fungus Fusarium is of concern to agriculture because it can cause crop diseases and produce mycotoxins, including three (fumonisins, trichothecenes, and zearalenone) that are among the mycotoxins of greatest concern to food and feed safety. Mycotoxin contamination and crop diseases caused by Fusarium result from a combination of factors, including species of Fusarium, crop species/cultivar, other microbes, and the environment. We will use multiple approaches to identify critical components of Fusarium biology that contribute to crop diseases and mycotoxin contamination, with an emphasis on fumonisins produced by Fusarium verticillioides. We will use genomics to identify genetic markers that provide an unprecedented ability to identify diverse Fusarium species and to resolve phylogenetic relationships among species. We will also use genomics to elucidate the genetic potential of diverse Fusarium species to produce mycotoxins as well as the genetic mechanisms that affect distribution of mycotoxin biosynthetic genes. In addition, we will use mutagenesis to identify genes that suppress fumonisin production in F. verticillioides. Interactions of Fusarium and crops that lead to mycotoxin contamination likely result, in part, from metabolites produced by each organism. Thus, we will use mass spectrometry-based metabolomics to identify metabolites formed during the interaction of F. verticillioides and maize to determine which metabolites are critical for fumonisin contamination. We will also employ a transcriptomics approach to elucidate the effects of one class of plant metabolites, oxylipins, on fumonisin production in F. verticillioides. Because Fusarium mycotoxin levels are typically higher in crops with high levels of Fusarium-incited diseases, improving crop disease resistance will likely reduce mycotoxin contamination as well. Plant chitinases are enzymes that degrade chitin, an essential component of fungal cell walls, and likely contribute to fungal disease resistance. To elucidate how chitinases can be manipulated to improve this resistance, we will use proteomics to study the interaction of maize chitinases and fungal proteases that inactivate chitinases. We will also use classical mycological methods and DNA-based phylogenetic analyses to evaluate the range of fungal endophytes that occur in maize under diverse environmental conditions and to identify endophytes that can inhibit growth and/or fumonisin production in F. verticillioides. We will also use transcriptomics to determine the mechanism by which the fungal endophyte Talaromyces inhibits F. verticillioides. Finally, we will use quantitative polymerase chain reaction (PCR) to determine whether mycotoxin contamination contributes to the ability of F. verticillioides to compete with other maize-associated fungi.
Progress Report
Objective 1: Species of 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
1. 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.
2. 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.
3. 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.
Review 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. https://doi.org/10.1016/j.fgb.2018.11.001.
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. https://doi.org/10.3389/fpls.2018.01936.
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. https://doi.org/10.1016/j.pmpp.2018.12.004.
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. http://dx.doi.org/10.1021/acs.jafc.8b05564.
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. https://doi.org/10.1094/PDIS-01-17-0103-RE.
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. https://doi.org/10.1080/00275514.2018.1526619.
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. https://doi.org/10.1080/00275514.2018.1519773.
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. https://doi.org/10.1186/s12864-019-5567-7.
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. https://doi.org/10.1080/00275514.2018.1512296.
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. https://doi.org/10.3389/fmicb.2019.01247.
