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Research Project: Eliminating Fusarium Mycotoxin Contamination of Corn by Targeting Fungal Mechanisms and Adaptations Conferring Fitness in Corn and Toxicology and Toxinology Studies of Mycotoxins

Location: Toxicology & Mycotoxin Research

2021 Annual Report

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 hostinduced 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 cocontaminated corn using an in vivo rodent bioassay approach incorporating biomarkers.

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
The unit operated under bridge project #6040-42000-045-000D in FY2021 due to a 1-year postponement of our research review through the Office of Scientific Quality Review. This postponement was granted to provide needed time to hire new scientists and technical staff and to reformulate the mission and objectives of the unit’s new research direction, which is focused on mitigating mycotoxins in poultry feed. In the past year the unit onboarded two new scientists, one support microbiologist, four technicians, and a new Research Leader. The Unit also increased its outreach and interactions with stakeholders aligned with the new mission. In this regard FY2021 was a very productive and successful year. In terms of research, the information presented below is primarily a five-year summary of the expired project #6040-42000-043-000D (2016–2021) since we did not have defined objectives directly associated with our bridge project. Objective 1: Soilborne plant pathogenic fungi are well-adapted to their varied environments. 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 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 another corn-infecting fungus, Sarocladium zeae, can shut down the production of fumonisin mycotoxins by F. verticillioides. This discovery by ARS scientists has great potential as a novel control strategy for mitigating fumonisin contamination of corn. It is also a textbook example of the impact microbial metabolites can have on the biology of other microbes. Regarding microbial interactions, a modified biosensor system was utilized by ARS scientists 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, ARS scientists 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 mechanistically studied in detail to evaluate their role in the production of nitrous oxide, a major greenhouse gas, and the ability of the fungi to grow under low oxygen conditions. 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 be tested as candidate inhibitors of fungal denitrification. By successfully inhibiting this physiological process, agricultural emissions of nitrous oxide may be reduced and the survival and fitness of mycotoxin producing fungi may be reduced. 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. ARS scientists hypothesize 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 regarding the use 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. Similar growth inhibition of A. flavus was observed. 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). ARS scientists contracted with a fee-for-service facility at the University of Wisconsin to generate transgenic corn lines designed to silence the essential fungal genes. The transgenic seed were screened using a traditional seedling disease assay, which proved inconclusive. A subsequent collaborative project to evaluate whether the transgenic corn is resistant to ear-rot was delayed due to the pandemic but will be revisited. Transgenic lines demonstrating resistance will be grown to amplify seed for further evaluation. Objectives 4 and 5: These two objectives were inherited 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 ahead of schedule in FY2019.

1. Poultry processing sanitation procedures may be causing unintentional, incidental production of a banned chemical compound on poultry meat. Detection of semicarbazide (SEM) in poultry products is a serious problem for U.S. producers. The presence of SEM in meat is problematic since the compound is considered an indicator for use of nitrofurazone, a banned antibiotic. Recently the detection of SEM caused trade disputes with a significant importer of U.S. poultry, resulting in an import ban that “delists” specific processing plants. However, evidence has emerged that sanitizers used in processing facilities can chemically create SEM from biological molecules in the absence of nitrofurazone use. Validation studies conducted by ARS researchers in Athens, Georgia, support this unintentional production of SEM on poultry meat. Substantial progress was made toward method development for the analysis of SEM in chicken meat, as well as new data on the mechanistic details of incidental SEM production. Additionally, a large survey of poultry processing plants, including some of the delisted plants noted above, was conducted this year. The survey indicated the use of certain antiseptic chemicals, in combination with pH, can react with meat tissue to produce detectable levels of SEM, thus confirming that incidental production of the chemical can occur in processing facilities. This is further evidence that SEM should not be used as an indicator of nitrofurazone use. This work may result in improved sanitation procedures that prevent the unintentional production of SEM, thus saving the U.S. poultry industry millions of dollars annually by avoiding future trade disputes