Location: Toxicology & Mycotoxin Research
2020 Annual Report
Objectives
1. Determine the evolutionary history and molecular genetics of metabolic and developmental features enhancing the fitness of mycotoxigenic Fusarium (F.) species, including such areas as xenobiotic tolerance, denitrification, and nitric oxide detoxification and the contribution to greenhouse gas emission.
1.1. Identify and characterize hydrolytic lactamases conferring adaptive advantages to F. verticillioides.
1.2. Determine if F. verticillioides produces quorum sensing or quorum sensing inhibitory compounds in vitro and during endophytic colonization of corn.
1.3. Evaluate denitrification by Fusarium species and its impact on competitive fitness, in planta production of mycotoxins, and the production of the potent greenhouse gas, nitrous oxide (N2O).
2. Evaluate the influence of a common niche on the evolution and adaptation of two co-occurring, seed-borne, metabolically active maize endophytes, Acremonium (A.) zeae and Fusarium (F.) verticillioides.
2.1. Utilize comparative genomics to determine if F. verticillioides and A. zeae share gene clusters or other features that correlate to corn as their common host.
2.2. Evaluate competitive interactions between F. verticillioides and A. zeae and profile their transcriptional and metabolic responses.
3. Develop and improve control strategies for mycotoxin contamination by targeting fungal-specific enzymatic activities, using molecular technologies such as host-induced gene silencing.
3.1. Develop and express RNAi silencing constructs for in vitro growth inhibition of F. verticillioides.
3.2. Develop and transform into corn functional vector(s) for host-induced gene silencing (HIGS).
3.3. Testing transgenic corn lines for resistance to F. verticillioides.
4. Determine the interactions between fumonisin exposure and dietary factors on fetal and postnatal development using animal models to provide basic information for ongoing translational human studies.
5. Determine the efficacy of cooking methods to detoxify mycotoxins in co-contaminated corn using an in vivo rodent bioassay approach incorporating biomarkers.
Approach
1. Lactamase genes in Fusarium (F.) verticillioides confer resistance to environmental lactam-containing antibiotic compounds. F. verticillioides metabolites impact quorum sensing related activities. Fusarium species, notably F. verticillioides, have an active denitrification pathway that is linked to nitric oxide detoxification.
2. Association of F. verticillioides and Acremonium (A.) zeae with the common host (corn) resulted in the two fungi sharing highly homologous genes or gene clusters. F. verticillioides and A. zeae antagonistically interact with distinct transcriptional and metabolic reprogramming.
3. Construct silencing vectors, express in vitro, and conduct assays exposing F. verticillioides to the RNAi transcripts. Silencing constructs having in vitro efficacy will be transformed into corn. Lines of transformed corn will be screened for reduced infection, disease, and fumonisin accumulation.
4. Compare dose-response in mouse strains sensitive (LM/Bc) and insensitive (SWV) to neural tube defect induction by fumonisin B1. Compare dose-response for neural tube defect induction and selected gene expressions in fumonisin B1-exposed mice fed folate deficient or folate sufficient diets. Compare neonatal growth rates in offspring of mice fed diets containing fumonisins.
5. Determine the efficacy of alkaline cooking (nixtamalization) to detoxify corn
contaminated with aflatoxin alone or co-contaminated with aflatoxin and fumonisin
using a rat feeding bioassay.
Progress Report
Objective 1: Soilborne plant pathogenic fungi are well-adapted to their environments, which include their host(s) and the soil in which they reside. These fungi have developed complex metabolic strategies for competition, survival, and proliferation. For example, Fusarium verticillioides possesses an abundance of genes that are hypothesized to confer tolerance to antifungal compounds produced by the host plant (corn) or more frequently to competitor microbes in the cornfield soil environment. Fungi inhabiting less-complex environments possess fewer representatives of these genes. This research aids the identification of potential antifungal treatments. Additionally, we discovered that sub-inhibitory amounts of pyrrocidines, which are metabolites produced by Sarocladium zeae, can shut down the production of fumonisin mycotoxins by F. verticillioides. Sarocladium zeae is another fungus infecting corn. This discovery by ARS scientists has great potential as a novel control strategy for mitigating fumonisin contamination of corn.
Regarding microbial interactions, a modified biosensor system was utilized by ARS researchers to demonstrate that mycotoxins and other fungal secondary metabolites can inhibit bacterial quorum sensing, thus limiting the growth and other biological features of the bacteria. We now have greater understanding of how F. verticillioides may modulate bacterial populations.
Further, we are investigating fungal denitrification, another unique physiological activity of Fusarium species found only in a few other fungal groups. The F. verticillioides genes conferring denitrification have been identified and are being deleted so that the mutants can be functionally studied to experimentally assess the role of the genes in the physiological process of hypoxia-induced nitrate respiration and production of nitrous oxide. A Headquarters-funded Research Associate and a PhD student from the University of Georgia are focusing on this project. Utilizing an in silico computational approach, ARS scientists screened nearly 500,000 small compounds and identified 25 with high binding affinities to predicted structures of F. verticillioides denitrification enzymes. These compounds will next be tested as candidate inhibitors of fungal denitrification.
Objective 2: The genomes of multiple strains of Sarocladium zeae (formerly Acremonium zeae) were sequenced to enhance our ability to identify common genes shared by S. zeae and F. verticillioides since both fungi commonly infect corn kernels. Our hypothesis is that corn, as a common host, is a driving factor in the evolution of these fungal genomes. We have successfully determined that S. zeae possesses a gene cluster that is evolutionarily related to the same cluster of genes in F. verticillioides. One of the cluster genes (MBL1) in F. verticillioides was shown to confer resistance to antimicrobial chemicals produced by corn such as 2-benzoxazolinone (BOA). Using a CRISPR-Cas9 approach for gene editing in S. zeae, ARS has shown that the same gene in S. zeae also confers resistance to BOA. Further, we have analyzed S. zeae genomes for secondary metabolite gene clusters with the goal of identifying the genes responsible for biosynthesis of the pyrrocidine metabolites. Further, competitive interactions between F. verticillioides and S. zeae are being studied, particularly with regard to using S. zeae as a biocontrol agent to reduce both the fumonisin contamination of corn kernels and the infection of kernels by F. verticillioides. Competition assays have indicated S. zeae is also producing a metabolite other than pyrrocidine responsible for inhibiting the growth of F. verticillioides. Additionally, to determine the benefit of S. zeae as a biocontrol agent against other fungi, we also conducted competition assays involving Aspergillus flavus, the primary producer of the carcinogenic aflatoxin mycotoxin. The same effect of growth inhibition seen with F. verticillioides by S. zeae was also shown to occur with A. flavus.
Objective 3: Three genes of F. verticillioides essential for growth and development are being targeted for “silencing” by a strategy called host-induced gene silencing (HIGS). We contracted with a fee-for-service facility at the University of Wisconsin to generate transgenic corn lines. We received more than expected quantities of both transgenic events and the amount of seed per event. We could therefore forego the amplification of seed expected at this stage of the project. We instead carried out the intended multiple seedling disease experiments and identified several 1:1 segregating transgenic lines. After extensively replicated seedling disease assays, we did not conclusively demonstrate any resistance effect conferred by the transgene. This is likely an artifact of the assay. We will next conduct ear-rot assays to determine the level of resistance to F. verticillioides conferred by HIGS. For those experiments we currently have sufficient seed. Transgenic lines demonstrating resistance will then be grown to amplify seed.
Objectives 4 and 5: These two objectives are from former Project No. 6040-42000-013-00D and were consolidated into the active project reported herein. Due to the retirement of the responsible scientists and resulting vacancies, these objectives were terminated in FY2019.
Accomplishments
1. Unintentional production of chemicals on poultry meat due to sanitation procedures. The detection of semicarbazide (SEM) in poultry products has caused a trade dispute with a significant importer of U.S. processed meat, resulting in an import ban that “delists” specific processing plants. The presence of SEM in meat is considered to be indicative of nitrofurazone use, a banned antibiotic. However, evidence has emerged that sanitizers used in food processing can chemically create SEM from biological molecules in the absence of nitrofurazone use. Validation studies conducted by ARS researchers in Athens, Georgia, so far support this unintentional production of SEM on poultry meat. Substantial progress was made toward method development for the analysis of SEM in chicken, as well as preliminary results in the mechanistic investigation. Additional studies will be performed to increase accuracy of the analyses and to establish evidence of reaction mechanisms for SEM production. This work will culminate in a large survey of poultry processing plants, both banned and otherwise, in an attempt to correlate SEM production with the processing and sanitation conditions in those plants. The outcome of this work will be revised sanitation procedures that prevent the unintentional production of SEM and hopefully a resolution to the trade dispute.
2. A battle between fungi infecting corn kernels. Fungi such as Fusarium verticillioides are major concerns for food safety because of the mycotoxins they produce, most notably the fumonisins that cause a number of species-specific animal diseases and may cause neural tube birth defects, stunting, and esophageal cancer in humans. Control of the fungus and preventing the contamination of food and feed with fumonisins are difficult goals to achieve for various reasons. Further, studies suggest fumonisin results in $20 million to $200 million in losses annually to U.S. corn producers. ARS researchers in Athens, Georgia, have discovered a group of chemical compounds that inhibit the production of fumonisins by F. verticillioides. These are the pyrrocidines produced by another corn kernel inhabiting fungus, Sarocladium zeae. This fungus does not cause any plant disease symptoms and does not produce any known mycotoxins. The potential use of pyrrocidines as inhibitors of fumonisin production could have immense impact on the safety and value of corn and its processed products by reducing and managing fumonisin contamination. This would be a significant contribution to food safety and provide economic relief to corn growers and processors.
Review Publications
Rath, M., Crenshaw, N.J., Lofton, L., Glenn, A.E., Gold, S.E. 2020. FVSTUA is a Key Regulator of Sporulation, Toxin Synthesis and Virulence in Fusarium verticillioides. Molecular Plant-Microbe Interactions. 33:958-971. https://doi.org/10.1094/MPMI-09-19-0271-R.
Gao, M., Glenn, A.E., Gu, X., Mitchell, T.R., Satterlee, T., Duke, M.V., Scheffler, B.E., Gold, S.E. 2020. Pyrrocidine, a molecular off switch for fumonisin biosynthesis. PLoS Pathogens. 6;16(7):e1008595. https://doi.org/10.1371/journal.ppat.1008595.
