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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Toxicology & Mycotoxin Research » Research » Research Project #430468

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

2018 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
Plant pathogenic fungi are incredibly well-adapted to their hosts and the general environments in which they reside, and many of these fungi have developed complex metabolic strategies for competition, survival, and proliferation. For example, Fusarium verticillioides possesses 46 genes putatively encoding lactamases that may confer tolerance to antifungal compounds produced by the host plant (corn) or generally encountered in the cornfield environment. Using a gene deletion strategy developed by the Unit, we have created deletion mutants for most of these lactamase genes, and this library of mutants has allowed us to test various lactam compounds for their antifungal effects and identify what genes confer resistance to the lactams. This research is allowing us to identify potential antifungal treatments. Further, gene expression data resulting from exposure of F. verticillioides to various lactam compounds, including pyrrocidines, the antibiotic compounds produced by Acremonium zeae, have resulted in identification of novel genes for further functional characterization. We have not created double or higher order gene deletion mutants since they are not yet needed. However, we will soon complete a series of backcrosses resulting in the creation of a new strain that will be nearly isogenic with our current wild-type strain except for a necessary genetic difference allowing the two strains to mate. This is useful to reduce genetic noise in progeny resulting from sexual crosses. The result will be cleaner, more predictive, and more scientifically sound genotypes when making double or higher order gene deletion mutants. In terms of microbial interactions, we have utilized a modified biosensor system to show that mycotoxins and other fungal secondary metabolites can inhibit bacterial quorum sensing, thus limiting the growth and other biological features of the bacteria. Quorum sensing is a microbial mechanism used to synchronize all physiological activities within a population in order to enhance the ecological fitness abilities of that organism, and 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 deleted, and the mutants are being functionally studied to experimentally assess the role of the genes in the physiological process of hypoxia-induced nitrate respiration and production of nitrous oxide. The instrumentation for measuring nitrous oxide is now operational and initial testing is underway. We have developed the in vitro assays to phenotype and characterize the deletion mutants under various nutrient and oxygen conditions, including hypoxia (5% oxygen) using a custom-built chamber housing a Bioscreen C instrument for real-time, quantitative assessment of growth. We have demonstrated the F. verticillioides denitrification genes are highly induced in response to nitric oxide exposure even when oxygen levels are normal, indicating this pathway is not strictly related to hypoxic respiration but is also responsible for nitric oxide detoxification. Experiments to evaluate the impact of denitrification on interactions with corn plants have begun. Identification of inhibitors of denitrification have not fully begun, but we have continued to perfect the assay, and an ARS-funded research associate will soon be recruited to focus on this sub-objective. This study involves collaboration with the ARS Mycotoxin Prevention and Applied Microbiology Research Unit in Peoria, Illinois. The genomes of multiple strains of A. zeae were sequenced to enhance our ability to identify common genes shared by A. zeae and F. verticillioides. We have successfully determined that A. zeae possesses a gene cluster that is clearly syntenic with the same cluster of F. verticillioides. We have previously shown that this cluster was horizontally acquired by Colletotrichum graminicola from a progenitor of F. verticillioides. It now appears A. zeae is also part of the evolutionary history of this gene cluster. We have attempted to generate an A. zeae deletion mutant for the lactamase gene in this cluster, but to-date this has not been successful. To overcome this bottleneck, we are attempting to utilize a CRISPR-Cas approach for gene editing in A. zeae. Further, we have analyzed A. zeae genomes for secondary metabolite gene clusters with the goal of identifying candidate clusters responsible for biosynthesis of the pyrrocidines. Three clusters are good candidates and will be targeted for gene mutation studies and characterization with regard to pyrrocidine production. ARS researchers are hopeful a manuscript will be submitted within the next year. Lastly, competitive interactions between F. verticillioides and A. zeae are being studied, particularly with regard to using A. zeae as a biocontrol agent to reduce both the fumonisin contamination of corn kernels and the infection of kernels by F. verticillioides. Gene transcription and metabolite production data were generated for the interaction of these two fungi and will aid in our evaluation of A. zeae as a biocontrol strategy. Three genes of F. verticillioides essential for growth and development are being targeted for “silencing” by a strategy called RNAi. Unfortunately, our efforts to test this silencing in vitro have not succeeded so far. We consulted with others doing similar research and found this is a common problem. We are now investigating a new strategy for this in vitro proof-of-concept phase of the project. Another setback we encountered involved our collaborator, who no longer is able to generate and provide transgenic corn lines as was planned due to staffing issues. As a revised strategy, we have contracted with a fee-for-service facility at the University of Wisconsin for this work. We have also entered into a new collaboration with the ARS Feed and Food Safety Research Unit (New Orleans, LA) to develop transgenic corn for resistance to both F. verticillioides and Aspergillus flavus and their respective toxins, the fumonisins and aflatoxins.

4. Accomplishments
1. Fungal denitrification project recognized with the Translational Mycology Award from the Mycological Society of America. Bacteria have long been noted as the primary microbes responsible for denitrification, which is nitrate respiration under low oxygen resulting in release of atmospheric nitrogen. Yet, in the past few years studies have shown that fungi may actually be the primary denitrifying microbes, and instead of releasing nitrogen gas, fungi release the greenhouse gas nitrous oxide. Such emissions by fungi may explain agriculture’s significant contribution to global nitrous oxide levels. ARS researcher at Athens, Georgia, found that Fusarium species such as F. verticillioides are among a limited number of fungi having the full set of genes conferring denitrification, and F. verticillioides genes are highly expressed when the fungus is either grown under low oxygen (hypoxia) or exposed to nitric oxide. Further, nitric oxide induces these genes even when oxygen is available, indicating the pathway is not strictly a low oxygen respiration pathway but instead also functions to detoxify nitric oxide, which may enhance the virulence and environmental fitness of F. verticillioides. The ARS researchers are functionally characterizing these genes with the ultimate goal of identifying inhibitors of the encoded enzymes as a strategy to reduce nitrous oxide emissions and reduce the fitness of F. verticillioides. Such inhibition and reduced fitness should result in reduced production of fumonisin mycotoxins, thus resulting in corn that is safer for human and animal consumption. The Mycological Society of America recognized this unique and impactful research with its Translational Mycology Award for 2018.

Review Publications
Blacutt, A.A., Gold, S.E., Voss, K.A., Goa, M., and Glenn, A.E. 2018. Fusarium verticillioides: Fusarium verticillioides: Advancements in understanding the toxicity, virulence, and niche adaptations of a model mycotoxigenic pathogen of maize. Phytopathology.
Gao, S., Gold, S.E., Glenn, A.E. 2017. Characterization of two catalase-peroxidase-encoding genes in Fusarium verticillioides reveals differential responses to in vitro versus in planta oxidative challenges. Molecular Plant Pathology. doi:10.1111/mpp.12591.
Gao, M., Glenn, A.E., Blacutt, A.A., Gold, S.E. 2017. Fungal lactamases: Their occurrence and function. Frontiers in Microbiology. 8:1775. doi:10.3389/fmicb.2017.01775.
Bacon, C.W., Hinton, D.M., Mitchell, T.R., Palencia, E. 2018. In situ ergot alkaloid detection in three Balansia epichloe-infected grass species. Journal of Applied Microbiology. 10.1111/jam.13941.
Bacon, C.W., Hinton, D.M., Mitchell, T.R. 2018. Screening of Bacillus mojavensis biofilms and biosurfactants using laser ablation electrospray ionization mass spectroscopy. Journal of Applied Microbiology. 10.1111/JAM.13905.
Jogi, A., Kerry, J.W., Brenneman, T.B., Leebens-Mack, J.H., Gold, S.E. 2016. Identification of genes differentially expressed during early interactions between the stem rot fungus (Sclerotium rolfsii) and peanut (Arachis hypogaea) cultivars with increasing disease resistance levels. Microbiological Research. 184: 1-12. doi: 10.1016/j.micres.2015.11.003
Rath, M., Mitchell, T.R., Gold, S.E. 2018. Volatiles produced by Bacillus mojavensis RRC101 act as plant growth modulators and are strongly culture-dependent. Microbiological Research. 208:76-84.