Location: Mycotoxin Prevention and Applied Microbiology Research
2024 Annual Report
Objectives
Objective 1: Define diversity of mycotoxin-producing Fusarium species. [C1, PS1, PS2]
Sub-objective 1.A: Elucidate phylogenetic diversity, mycotoxin potential, and pathogenicity to cereals of fungi in the F. tricinctum species complex.
Sub-objective 1.B: Identify genomic and phenotypic differences in collections of F. proliferatum and F. verticillioides isolates to aid discovery of targets for control of fumonisins in corn.
Objective 2: Identify targets to reduce fumonisin contamination in corn. [C1, PS1, PS2, PS5]
Sub-objective 2.A: Determine whether the corn zmCRR1 protein contributes to resistance to fumonisin contamination.
Sub-objective 2.B: Identify corn genes encoding papain-like cysteine proteases involved in fumonisin contamination to aid genomics-assisted breeding.
Sub-objective 2.C: Reduce fumonisin contamination in corn by engineering kernel-specific expression of RNAi targeting the fumonisin biosynthetic gene FUM1.
Sub-objective 2.D: Determine how corn oxylipins control fumonisin production in F. verticillioides.
Sub-objective 2.E: Determine whether the killer meiotic drive element SkK can be used to drive biased transmission of a gene that blocks fumonisin production in F. verticillioides.
Approach
Fusarium species are fungi with potentially the greatest negative impact on agriculture. This is because of their collective abilities to produce mycotoxins and cause destructive diseases in crops, including the important cereals: corn, wheat, and rice. The Fusarium mycotoxins fumonisins and trichothecenes are among the mycotoxins of most concern to food and feed safety due to their toxicity and frequent occurrence in crops. However, Fusarium species produce other mycotoxins whose effects on food and feed safety are poorly understood. In the U.S., harmful impacts of mycotoxins on health are mitigated by removing contaminated grain from food/feed supply chains. Despite these efforts, however, the toxins continue to cause billions of dollars in agricultural losses. This project plan addresses knowledge gaps that hinder control of mycotoxins caused by two groups of Fusarium: the Fusarium tricinctum species complex (FTSC), which includes multiple species that cause head blight of small-grain cereals and produce multiple mycotoxins; and the F. fujikuroi species complex, specifically Fusarium proliferatum and Fusarium verticillioides, which are the primary causes of fumonisin contamination in corn. The proposed research has two objectives: i) define diversity of mycotoxin-producing Fusarium species, specifically members of the FTSC, F. proliferatum, and F. verticillioides; and ii) identify targets to reduce fumonisins in corn. To address the first objective, we propose to elucidate variation in genome sequences, mycotoxin production, and pathogenicity within and among Fusarium species. This will aid development of broadly effective control practices for Fusarium mycotoxins. To address the second objective, we propose to identify corn and Fusarium proteins/genes that can be used to enhance breeding or engineering strategies aimed at reducing fumonisin contamination. To address the second objective, we also propose to develop fumonisin reduction methods based on two biological phenomena: RNA interference and meiotic drive elements. The research accomplishments will aid efforts to reduce mycotoxin contamination in corn and other cereal crops, and will benefit growers, processors, regulatory agencies, and ultimately American consumers.
Progress Report
Objective 1: We have identified significant differences in the genetic diversity within the fungal species Fusarium proliferatum and F. verticillioides. These species are the predominant causes of fumonisin mycotoxin contamination in crops, including contamination in corn, which is one of the mycotoxin problems of greatest concern to food and feed safety worldwide. Using multi-step, computer-aided genetic analyses, we examined whole genome sequences (i.e., DNA sequences of all chromosomes) in 95 F. proliferatum strains and 113 F. verticillioides strains collected from multiple regions within the United States, elsewhere in North America, and in South America, Africa, Asia, and Europe. The results indicated that F. proliferatum is comprised of three genetically distinct groups. 80% of strains in one group are from the United States and southeastern Canada, while the other groups are mixtures of strains from most or all countries represented, including the United States. The results also indicated that although F. verticillioides did not consist of two or more distinct groups, the genetic diversity of F. verticillioides strains from the United States encompassed much of the genetic diversity that existed in strains from all other countries examined. Analysis of genes required for fumonisin production in F. proliferatum and F. verticillioides revealed that 5% of strains of each species have mutations that should block or significantly alter fumonisin production. We are currently examining the ability of strains to produce fumonisins as well as other characteristics that could impact the ability of the strains to cause fumonisin contamination in corn and other crops.
We also determined which species of the fungus Fusarium are present in corn kernels collected in 2021 from across the state of Illinois. This research was done as part of an effort to elucidate the cause(s) of higher levels of fumonisin contamination in southern Illinois, relative to other parts of the state. Using a combination of morphological and DNA-based methods, we determined that seven Fusarium species (F. andiyazi, F. fujikuroi, F. graminearum, F. poae, F. proliferatum, F. subglutinans, and F. verticillioides) were present with varying frequencies in the sampled kernels. The fumonisin-producing species F. verticillioides occurred more frequently in southern Illinois, where fumonisin contamination was more severe, than elsewhere in the state. We also identified three genetic variants of F. verticillioides and are conducting experiments to determine if the variants differ in fumonisin production and/or other agriculturally significant traits, pathogenicity, optimal growth temperature, carbon dioxide tolerance, and fungicide resistance.
We also investigated fungi and mycotoxins present prior to harvest in agricultural fields in 41 counties representing northern, central, and southern Illinois. The goal of this research is to aid development of ARS models that predict mycotoxin risk in Illinois. The models indicate that plant growth in March can predict post-harvest risk of fumonisin contamination in corn, even though corn is not planted until April or May, depending on the region of Illinois. To investigate how conditions prior to harvest impact post-harvest fumonisin contamination, we collected soil and plant debris from corn fields at three time points: 1) in March prior to planting, 2) in July at mid-season, and 3) in September prior to harvest. In September, we also sampled corn ears prior to harvest. Although fumonisin levels in the ears were relatively low in all Illinois counties sampled, the levels were higher in ears from southern Illinois than from central or northern Illinois.
We also continued to collaborate with researchers at the U.S. Department of Energy’s Joint Genome Institute (JGI) and the University of Massachusetts to develop a database of high-quality genome sequence data for fungal species that represent the breadth of genetic diversity of Fusarium. For this project, we prepared all RNA and DNA samples and shipped them to JGI for processing. JGI generated the genome data using long-read DNA sequencing technology. The resulting data provided theDNA sequences of full-length chromosomes of each species analyzed. Such high-quality genome sequences allow for analyses that are not possible with many other types of data. When data processing is completed for each species, the resulting sequence data are posted on JGI’s MycoCosm website. The site is an interactive database that allows the data to be analyzed, searched, and downloaded. According to JGI’s records, this collaboration has led to 9,503 unique users visiting the Fusarium pages at MycoCosm a total of 57,706 times.
Objective 2: We continue to investigate interactions between proteins produced by corn and by species of the fungus Fusarium that cause disease and toxin contamination in corn. We anticipate that understanding the details of such interactions will aid plant breeding and genetic engineering efforts to enhance resistance of corn to diseases and toxin contamination caused by Fusarium. The corn protein that we investigated can trigger immune responses in plants and is known as a papain-like protease. However, Fusarium species can produce proteins that inhibit the corn papain-like protease. To identify the corn papain-like protease, we used the predicted structures of papain-like proteases identified in other plants to search a database of predicted 3-dimensional structures of corn proteins that were generated by artificial intelligence. This search identified three potential corn papain-like proteases. Each of these proteins was produced in the yeast Pichia pastoris using standard microbial engineering methods. The proteins were then purified and are currently being assessed in biochemical assays to determine whether they are inactivated by a previously identified Fusarium protein that inactivates papain-like proteases.
We are also identifying and elucidating the function of bacterial proteins that improve plant health. Our approach is based on the idea that such proteins have potential to aid efforts to reduce crop diseases and the associated toxin contamination caused by fungi. We focused on bacteria that are members of the crop microbiome, that is, the community of microbes that live in, on and around crops. We have discovered a protein from a harmless soil bacterium that turns off the response that plants use to defend themselves from infection by pathogenic microbes. Shutting off the plant defense response allows the soil bacteria to grow near roots, which can enhance root health by, for example, promoting the growth of beneficial or harmless bacteria around the roots instead of pathogenic microbes. Identifying microbiome proteins that affect the plant defense response has potential to aid efforts to reduce fungal diseases and toxin contamination through manipulation of crop microbiomes.
Along with ARS collaborators in Ames, Iowa, we developed an online interactive database of predicted structures of all proteins produced by 22 species of the fungus Fusarium. The database is a research tool that aids identification of Fusarium proteins that affect the ability of the fungus to infect crops, cause disease, and/or produce toxins that are health hazards. Such proteins are potential targets in efforts to reduce diseases and toxin contamination in crops caused by Fusarium. The utility and novelty of the database is its interactive nature, which allows users to investigate predicted structures and functions of the proteins, including how they could contribute to the ability of Fusarium to infect crops. The database also allows users to investigate whether variants of the same protein are likely to impact how the protein functions.
We continued to investigate the potential to use the Fusarium verticillioides gene SKC1 to reduce fumonisin contamination caused by the fungus. Previously, we showed that although SKC1 can disperse itself among isolates of F. verticillioides via the sexual cycle of the fungus, it could not disperse itself when fused to a gene designed to block fumonisin production. Recently, we found evidence that SKC1 dispersal might require two other F. verticillioides genes in addition to SKC1. Experiments are in progress to confirm that these two genes impact SKC1 dispersal.
We also investigated whether the fungus Trichoderma harzianum and the bacterium Bacillus can reduce fumonisin contamination in corn. A private company has identified strains of T. harzianum and Bacillus that can enhance tolerance of corn to heat and drought stress. Because fumonisin contamination in corn is associated with these stresses, the two microbes have potential to reduce fumonisin contamination. In FY 2024, we began a two-year study to investigate this possibility. We applied the microbes by seed treatment (T. harzianum only) and foliar spray (both microbes). Subsequently, we infected the treated corn with the fumonisin- producing fungus F. verticillioides using standard methods that cause fumonisin contamination. Analysis of fumonisin contamination in the resulting corn did not provide evidence that T. harzianum or Bacillus reduced contamination, but this could have been due to highly variable fumonisin levels in the corn. Further study is in progress.
Accomplishments
1. Novel approach to detect toxins in crops. Fumonisins are a group of fungal toxins that frequently contaminate corn and pose health hazards to humans, livestock, and pets. As a result, fumonisins contribute to the estimated losses of $0.5 – 5 billion to U.S. and Canadian agriculture caused by fungal toxins each year. Additional tools are needed to improve the accuracy and efficiency and reduce costs of detecting the toxins and toxin-producing fungi in crops. Metabolites that fungi emit as volatiles (i.e., gasses) have potential as a method of detecting toxins and toxin-producing fungi. Therefore, researchers at Liege University in Belgium, the National Research Council in Italy, and ARS researchers in Peoria, Illinois, examined production of volatile metabolites produced by the fungus Fusarium, which is the predominant cause of fumonisin contamination in corn. The researchers identified volatile metabolites that were produced by fumonisin-producing strains of Fusarium but not by toxin-nonproducing strains. These results provide fundamental information that can be used by government, academic, and private-sector researchers to develop novel tools to detect toxin contamination in crops.
2. Identified a fungal protein that inactivates a corn defense protein. Fungi can infect corn in the field, cause disease, and contaminate grain with toxins that pose health hazards. The diseases and toxin contamination can cause hundreds of millions of dollars in losses to U.S. agriculture each year. Therefore, ARS researchers in Peoria, Illinois, and at the University of Waterloo in Canada are searching for novel strategies to control the diseases and toxin contamination by investigating how interactions of corn and fungal proteins impact disease development. The researchers targeted a fungal protein, polyglycine hydrolase, that degrades a corn defense protein called chitinase. A combination of laboratory experimentation, computational analyses, and computer modeling were used to determine the structure and function of polyglycine hydrolase. The researchers found that the protein has two distinct parts. One part resembled bacterial proteins that inactivate antibiotics. The other part is novel to science but might help guide the protein to the target site in corn. These discoveries will aid plant breeding programs aimed at enhancing resistance of corn to fungal diseases. This, in turn, will improve quality and safety of corn and reduce economic losses caused by fungi.
3. Developed an artificial intelligence approach to improve safety and quality of corn. Corn is the most important U.S. crop in terms of total production with an average value of $79.5 billion over the past four years. Quality, safety, and yield of corn are negatively impacted by fungal diseases and toxins as well as adverse weather. Understanding how changes in corn proteins affect their functions are necessary to improve resistance of this important crop to diseases, toxin contamination, and adverse weather. Therefore, ARS researchers in Peoria, Illinois, and Ames, Iowa, used artificial intelligence to develop an interactive online database, called PanEffect, that allows plant breeders and other scientists to investigate how variation in corn proteins affect their functions. The PanEffect database is hosted by the Maize Genetics and Genomics Database and includes information on approximately 40,000 proteins from 50 different types of corn. The database allows researchers to investigate the functions of proteins using computer modeling, and then use the resulting information to guide laboratory experiments, including breeding efforts and genetic engineering. Thus, the PanEffect database will allow scientists to identify variation in proteins that can be used to enhance resistance of corn to disease, toxin contamination, and adverse weather with greater efficiency and accuracy.
4. Identification of agricultural practices to limit toxin contamination in corn. Corn is a major cereal crop that contributes to food security and self-sufficiency in Ethiopia. One of the major limitations of corn production is Fusarium ear rot (FER), which is caused predominantly by the fungus Fusarium verticillioides. This and other FER fungi can also contaminate corn kernels with toxins that are a health hazard. ARS researchers in Peoria, Illinois, and at Haramaya University and Jimma University in Ethiopia, and at the International Maize and Wheat Improvement Center (CIMMYT) in Kenya, conducted a two-year (2020-2021) survey of 480 farms in 20 Ethiopian districts to determine the levels of FER at each location and to determine whether agronomic practices are associated with the disease. The research revealed that FER was widespread in Ethiopia, and districts with higher levels of the disease had the highest crop yield losses. High levels of insect and weed infestation, low nitrogen fertilizer application, and low frequency of insecticide spraying were associated with higher levels of FER. Moreover, agricultural practices such as early planting, three-round tillage, crop rotation, and inter-cropping systems were associated with lower levels of FER and higher yields. In addition, corn harvested from locations with more FER contained higher levels of the toxins fumonisins. This research enhances the global understanding of the Fusarium-corn-environment interactions. This understanding will aid surveillance and detection efforts to prevent diseases and toxin contamination of crops caused by exotic plant pathogens.
Review Publications
Deressa, T., Girma, A., Suresh, L.M., Bekeko, Z., Opoku, J., Vaughan, M., Proctor, R.H., Busman, M., Burgueno, J., Prasanna, B.M. 2023. Biophysical factors and agronomic practices associated with Fusarium ear rot and fumonisin contamination of maize in multiple agroecosystems in Ethiopia. Crop Science. https://doi.org/10.1002/csc2.21159.
Josselin, L., Proctor, R.H., Lippolis, V., Cervellieri, S., Hoylaerts, J., De Clerck, C., Fauconnier, M.-L., Moretti, A. 2023. Does alteration of fumonisin production in Fusarium verticillioides lead to volatolome variation? Food Chemistry. 438. Article 138004. https://doi.org/10.1016/j.foodchem.2023.138004.
Naumann, T.A., Dowling, N.V., Price, N.P., Rose, D.R. 2024. In vitro functional analysis and in silico structural modelling of pathogen-secreted polyglycine hydrolases. Biochemical and Biophysical Research Communications. https://doi.org/10.1016/j.bbrc.2024.149746.
McMillan, S.D., Oberlie, N.R., Hardtke, H.A., Montes, M.M., Brown, D.W., McQuade, K.L. 2023. A secondary function of trehalose-6-phosphate synthase is required for resistance to oxidative and desiccation stress in Fusarium verticillioides. Fungal Biology. 127(3):918-926. https://doi.org/10.1016/j.funbio.2023.01.006.
Andorf, C.M., Haley, O., Hayford, R.K., Portwood II, J.L., Harding, S.F., Sen, S., Cannon, E.K., Gardiner, J.M., Kim, H., Woodhouse, M.R. 2024. PanEffect: a pan-genome visualization tool for variant effects in maize. Bioinformatics. 40(2). Article btae073. https://doi.org/10.1093/bioinformatics/btae073.
Jeong, E., Lim, J.Y., Proctor, R.H., Lee, Y.-W., Xu, J., Shi, J., Liu, X., Seo, J.-A. 2023. Genome sequence resource of the head blight pathogens Fusarium asiaticum and F. graminearum isolated from cereal crops and gramineous weeds in Korea and China. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-10-22-0120-A.
Rogers, A., Jaiswal, N., Roggenkamp, E., Kim, H., MacCready, J.S., Chilvers, M.I., Scofield, S.R., Iyer-Pascuzzi, A.S., Helm, M.D. 2024. Genome-informed trophic classification and functional characterization of virulence proteins from the maize tar spot pathogen Phyllachora maydis. Phytopathology. https://doi.org/10.1094/PHYTO-01-24-0037-R.