MYCOTOXINS: BIOSECURITY, FOOD SAFETY AND BIOFUELS BYPRODUCTS (NC129, NC1025)

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0223848
• Project No.: ARK02300
• Multistate No.: NC-_OLD1183
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2015

Project Director
Bluhm, B.

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
Plant Pathology

Non Technical Summary
Preventing mycotoxin contamination of food and feed, and reducing the deleterious effects of mycotoxins on livestock, are important issues facing grain and livestock producers. Mycotoxins in grains processed for biofuels become concentrated in the solid byproduct known as distillers grain (DG), an important source of supplemental income for biofuel producers, and mycotoxin contamination is a major concern. For grain buyers and food processors, the primary need is a reliable method for rapid assessment of grain quality pertaining to mycotoxins and mycotoxigenic fungi. The worst-case scenario for these stakeholders is to own millions of bushels of corn contaminated with unacceptable levels of aflatoxins and fumonisins, or of wheat with excessive concentrations of deoxynivalenol (DON). Additionally, these stakeholders need cost-effective methods to predict, monitor, and minimize mycotoxin production in the field, and to detoxify mycotoxins and prevent further deterioration in contaminated grain. The lowering of tolerance limits for mycotoxins in overseas markets has increased the burden for grain buyers and food processors; currently, levels of mycotoxins that are acceptable for some US products are unacceptable in European and Asian markets, resulting in non-tariff trade barriers. Finally, workers who are responsible for animal and human health need information about the toxicity, carcinogenicity, modes of action, and biomarkers of exposure and disease for all categories of mycotoxins. To address these needs, the objectives of this project are to 1) Develop data for use in risk assessment of mycotoxins in human and animal health, 2) Establish integrated strategies to manage and to reduce mycotoxin contamination in cereal grains and distillers grains, and 3) Define the regulation of mycotoxin biosynthesis and the molecular relationships among mycotoxigenic fungi. Methods to be used include evaluating structure activity relationships (SAR) and investigating cell, tissue and whole animal responses at the biochemical, physiological and structural levels, creating molecular techniques to develop methods to monitor mycotoxigenic fungi and test new and old technologies designed to destroy mycotoxins monitor and predict their production in the field, and functional genomics. The outcomes derived from the work outlined in this proposal will contribute towards the overall goals of the National Institute of Food and Agriculture (NIFA). Results will have a major impact on government decision-making by providing a better understanding of how various environmental and food processing components affect mycotoxin biosynthesis, advance detection technologies that can be used by public agencies, and by private diagnostic labs to provide mycotoxin analysis services to food industries, and create new basic knowledge that can be incorporated into new management strategies to help grain growers minimize mycotoxin contamination.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4020	1040	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1040 - Molecular biology;

Keywords

mycotoxins

ear rot

head blight

management

pathogen detection

distillers grains

Goals / Objectives
Objectives: #3 Define the regulation of mycotoxin biosynthesis and the molecular relationships among mycotoxigenic fungi Outputs: Refereed journal publications; many will be co-authored by the members from multiple states. Development and validation of new management tools for diagnosis and prevention of mycotoxin contamination. Transfer of valuable research information to clientele groups (industry, government, grain producers and food producers) through general publications, website, and extension programs.

Project Methods
Objective 1. Define the regulation of mycotoxin biosynthesis and the molecular relationships between mycotoxigenic fungi Grains are a major source of food and energy for the world s population. Mycotoxigenic fungi are present in essentially every agricultural field, and mycotoxin contamination represents one of the greatest continuing threats to food safety and profitability. Mycotoxin contamination of grains that enter the food, feed, and ethanol industries is estimated to cause over $100 million in losses annually. The goal of this proposal is to better understand the molecular and genetic systems that link fungal development, pathogenicity and mycotoxin production. Members of our committee have formed collaborative teams based on specific mycotoxins and fungal species.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience: Target groups include corn growers in Arkansas and other states in the southern U.S., as well as the international mycotoxin research community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities for graduate students, post-doctoral assocites, and undergraduate researchers. How have the results been disseminated to communities of interest?Results have been disseminated through peer-reviewed publications, internet-based media, presentations at grower meetings, and directly to growers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? BDM1 of Fusarium graminearum was identified and characterized. This gene regulates pathogenesis during infection ofwheat and maize. It also regulates the biosynthesis of deoxynivalenol, an important trichothecene mycotoxin. FUG1 of Fusariumverticillioides was identified. Functional characterization of this novel gene suggests it represents a new class of fungal transcription factors. FUG1 regulated maize kernel infection and fumonisin biosynthesis. Interestingly, FUG1 also is required for defense against anti-microbial compounds produced during the maize defense reaction. The HAP regulatory complex ofFusariumverticillioideswas functionally characterized. A key component of the complex, HAP3, was deleted and mutants were characterized. The HAP complex is required for pathogenesis of corn kernels and stalks. Importantly, the HAP complex is also required for fumonisin biosynthesis. A genome sequence was obtained for Stenocarpella maydis, cause of Diplodia ear rot of corn. After functional annotation of predicted genes, emphasis was placed on identifying secondary metabolite clusters, with particular focus on clusters associated with mycotoxin production. Surveys of fungi associated with head mold in Arkansas indicated that sorghum grain was consistently infected with a range of Fusarium species, many of which were capable of producing various mycotoxins. Interestingly, a majority of the Fusarium fungi identified were F. equisiti (or close relatives), which produce a suite of mycotoxins, some of which have not yet drawn widespread public notice. Information derived from this project has been used to create experimental plant transgenes that target mycotoxin biosynthesis. Transgenic corn plants have been created and propigatged. Testing the efficacy of the transgenes will be beyond the scope of the current project'sreporting period.

Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Horevaj P, and Bluhm BH. 2012. BDM1 of Fusarium graminearum regulates virulence during infection of wheat and maize. Molecular Plant Pathology DOI: 10.1111/j.1364-3703.2011.00758.x.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Ridenour J, Bluhm BH. 201-. The novel fungal-specific gene FUG1 has a role in pathogenicity and fumonisin biosynthesis in Fusarium verticillioides. Molecular Plant Pathology
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Ridenour J, Smith J, Bluhm BH. 201-. The HAP complex of Fusarium verticillioides regulates fumonisin biosynthesis and kernel pathogenesis. Journal of Food Protection

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Target groups include corn growers in Arkansas and other states in the southern U.S., as well as the international mycotoxin research community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? PI Bluhm has further enhanced his knowledge and practical ability regarding bioinformatic analyses of fungal genome sequencing and assembly. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The genome sequence data for Stenocarpella maydis was refined with a more complete assembly. This work was performed in close collaboration with other members of the multi-state working group (Dr. Charles Woloshuk, PurdueUniversity; Dr. Lisa J. Vaillancourt, University of Kentucky). This work produced a genome assembly that is comparable to published de novo genome sequences in other filamentous fungal pathogens.

Publications

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Target groups include corn growers in Arkansas and other states in the southern U.S., as well as the international mycotoxin research community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? PI Bluhm and the graduate student involved in the project have both learned new skills and techniques related to bioinformatics. The graduate student received hands-on training in genomic DNA extraction, analysis, and sequencing as well. How have the results been disseminated to communities of interest? Pi Bluhm and the graduate student involved in the project traveled to Coker College (Hartsville, SC) to discuss the work to undergraduate students. Additionally, Bluhm and the graduate student participted in a DNA-based highschool out reach activity in Hartsville, SC. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A substanial amount of genome sequence data was created and analyzed for Stenocarpella maydis. This work was performed in close collaboration with other members of the multi-state working group (Dr. Charles Woloshuk, Purdue University;Dr. Lisa J. Vaillancourt, University of Kentucky). Genome sequencing and assembly has facilitated the identification of potential mycotoxin biosynthetic gene clusters in the pathogen. This information is the first genome sequence information available for this fungus. The Bluhm lab also worked to characterize an insertional mutant of the pathogen created in the Woloshuk lab. These accomplishments represent the first molecular analyses of mycotoxin biosynthesis in S. maydis.

Publications

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Efforts in Arkansas have focused on defining the regulation of mycotoxin biosynthesis and molecular relationships among mycotoxigenic fungi. To this end, we have been characterizing a CCAAT-binding, heterotrimeric transcription factor in Fusarium verticilliodes. Targeted disruption of any of the three genes encoding a core component yielded mutants with indistinguishable phenotypes, which included impairments in growth, conidiation, and pigmentation in culture. Pathogenesis on maize kernels and stalks was also substantially impaired. In addition to a reduction in growth, fumonisin levels were significantly lower than the wild-type or complemented strains after normalization to growth via ergosterol analysis. Homologs of these genes were deleted in Fusarium graminearum, and impairments in pathogenesis were observed. Phenotypic analyses of mutants in both species are ongoing. PARTICIPANTS: Dr. Burt Bluhm: PI Mr. John Ridenour: Graduate student (M.S.) Mr. Jon Smith: Graduate student (Ph.D.) Dr. Charles Woloshuk: Collaborator (Purdue University) Dr. Won-Bo Shim: Collaborator (Texas A&M University) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The identification of a novel molecular mechanism underlying Fusarium diseases of corn and fumonisin biosynthesis provides the scientific community important information for ongoing studies focused on the molecular dissection of mycotoxin contamination of grain.

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Research outputs of this project include the molecular dissection of pathways underlying mycotoxin biosynthesis in Fusarium graminearum during colonization of corn and wheat. Teaching activities related to these efforts include the advisement of a post-doctoral associate who performed most of the research. Additionally, research findings were presented to regional, national and international audiences at scientific meetings. PARTICIPANTS: Dr. Burt Bluhm: PI Dr. Peter Horevaj: Post-doctoral associate. Collaborating institution: Purdue University TARGET AUDIENCES: The target audience for this work is the scientific community studying plant-fungal interactions and mycotoxigenesis, particularly diseases caused by Fusarium graminearum. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Outcomes of this research include advancements in the fundamental understanding of how Fusarium graminearum interacts with its hosts at the molecular level, particularly with respect to the induction of mycotoxin biosynthesis. This new information can ultimately be incorporated into transgenic strategies to improve crop resistance.

Publications

• Horevaj P, and Bluhm BH. 2011. BDM1 of Fusarium graminearum regulates virulence during infection of wheat and maize. Molecular Plant Pathology DOI: 10.1111/j.1364-3703.2011.00758.x.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Consistent with Objective 3 (Define the regulation of mycotoxin biosynthesis and the molecular relationships among mycotoxigenic fungi), we surveyed grain sorghum in Arkansas to 1) identify and quantify mycotoxigenic fungi associated with head mold, and 2) to assess the mycotoxigenic potential of fungi isolated. We identified numerous mycotoxigenic Fusarium species, and determined that most isolates were capable of producing mycotoxins (i.e., fumonisins, trichothecenes) during colonization of sorghum heads. PARTICIPANTS: Participants included Dr. Burt Bluhm (PI), Dr. Peter Horevaj (Research associate), and Ms. Katie Crow (undergraduate student). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This activity contributed new knowledge regarding the potential threat of mycotoxin contamination of sorghum in Arkansas and identified specific mycotoxins that should be monitored in grain sorghum.

Publications

• No publications reported this period