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.
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.