Source: PURDUE UNIVERSITY submitted to
INTEGRATED MANAGEMENT STRATEGIES FOR ASPERGILLUS AND FUSARIUM EAR ROTS OF CORN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0230868
Grant No.
2013-68004-20359
Project No.
IND011264G2
Proposal No.
2012-02338
Multistate No.
(N/A)
Program Code
A5122
Project Start Date
Jan 1, 2013
Project End Date
Dec 31, 2018
Grant Year
2017
Project Director
Woloshuk, C.
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Botany & Plant Pathology
Non Technical Summary
US corn growers find it impossible to guarantee that their crop will contain acceptable levels of mycotoxins, such as aflatoxins and fumonisins. The tools currently available to manage mycotoxins are not consistently effective, and the risks associated with this uncertainty are poorly understood. The goal of this project is to develop resources that that will reduce the risk of aflatoxin and fumonisin contamination. The specific objectives are to optimize current management practices, develop the next generation of biocontrol agents, create novel sources of genetic resistance in corn, and develop decision tools that allow growers to calculate economic decision thresholds. Our study will involve field experiments and utilize model molecular technology. As a result of our research and extension efforts, we anticipate that corn producers will become more knowledgeable about how biocontrol agents impact the potential for mycotoxin contamination and be more aware of the management practices that can reduce the mycotoxin risks. Producers will apply biocontrol agents based on the population structure of the pathogen in the field and will use new information technology to assist them in management decisions. New knowledge is forthcoming about the molecular responses in corn to heat and water stresses. We anticipate the discovery of gene expression networks in in A. flavus and F. veticillioides that are associated with mycotoxin biosynthesis. There will be opportunities for product development including population structure assessments, design of biocontrol strains, real-time plant stress assessment, and corn hybrids expressing RNAi-transgenic technology. Finally, the research and educational opportunities that are part of our project will lead to more student who choose to pursue advanced degrees and careers in agriculture research.
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121510104020%
2121510110220%
2151510104025%
2151510110225%
6011510110210%
Knowledge Area
215 - Biological Control of Pests Affecting Plants; 212 - Pathogens and Nematodes Affecting Plants; 601 - Economics of Agricultural Production and Farm Management;

Subject Of Investigation
1510 - Corn;

Field Of Science
1102 - Mycology; 1040 - Molecular biology;
Goals / Objectives
The goal of this project is to provide growers with new control strategies and decision tools to reduce the risk of mycotoxin contamination in corn. Research and extension activities will address four overarching objectives: 1. Design and deliver best management practices for mycotoxin reduction; 2. Develop next generation of biological control; 3. Develop biomarkers for host susceptibility & generate RNAi-based transgenics for resistance; and 4. Assess risks, regulatory issues, and socioeconomic impacts Expected Outputs 1. Educational experiences (field days, workshops) for corn producers and grain handlers about ear rot/mycotoxin awareness and management 2. Print, video, web-based, and smartphone/electron tablet resources to aid producers in determining site-specific management practices to lower contamination 3. Optimized application recommendations for existing biocontrol products 4. Management practices developed for mycotoxin management in productions systems that use BT corn hybrids and foliar fungicides 5. A molecular marker protocol to determine the population structure for A. flavus in growers' fields, which will help assure selection of the most efficient biocontrol strains 6. A molecular marker protocol to determine the population structure for F. verticillioides in growers' fields 7. A defined management plan that, when implemented, will shift soil populations of A. flavus towards atoxigenic strains 8. A molecular marker protocol that will identify corn plants vulnerable to mycotoxin contamination because of environmental stress (an early warning system) 9. Novel transgenic corn lines that reduce the risk of mycotoxin contamination by RNAi-targeting of fungal genes involved in mycotoxin production 10. Computational tool that will enable network-based analysis of large-scale digital gene expression data 11. A benefit-cost analysis to corn growers for management strategies that lower aflatoxin and fumonisin contamination 12. An analysis that determines how the costs and benefits are distributed among the various stakeholders when management strategies that lower aflatoxin and fumonisin contamination are implemented 13. Research/extension experience for undergraduate students from Texas A&M Kingsville, a minority serving institution.
Project Methods
Replicated field trails in four states will evaluate current management practices, and optimize and validate new practices for controlling Aspergillus ear rot disease and aflatoxin contamination. Information from research will be distributed to stakeholders through newly developed print material, smartphone and electronic tablet applications, videos, web-based information, and through extension workshops. Molecular techniques will be used to characterize soil population structure of Aspergillus flavus and Fusarium verticillioides in Indiana, Arkansas, Texas, and North Carolina. Similar measures will be used to evaluate the impact of biocontrol agents on population structure of toxigenic A. flavus strains. We will measure phenotypic and molecular changes in corn under drought and heat stresses. From these data models will be developed to facilitate the selection of gene targets to control mycotoxin production as well as the identification of biomarkers for early warning of crop risk for mycotoxin contamination. Transgenic corn will be developed that targets specific genes associated with mycotoxin production. We will conduct cost analyses that will provide assessments of the benefits and risks of implementing the various management strategies developed in this project. A research mentoring program centered on the objectives of this grant will be developed for graduate and undergraduate students.

Progress 01/01/17 to 12/31/17

Outputs
Target Audience:Our project goals are 1) to provide corn growers with current best management practices for reducing ear rot diseases caused by the fungi Aspergillus flavus and Fusarium verticillioides and contamination with aflatoxins and fumonisins, respectively; and 2) to develop new management tools based on innovative genomic approaches. Our targeted audiences are corn producers, grain handlers, grain marketers, government officials, educators, and our scientific peers. Our communication methods include workshops, extension publications, websites, app, videos, peer reviewed publications, and oral/poster presentations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduates: Andrew Duncan - Research Assistant, North Carolina State University Eliza Enterline - Research Assistant, North Carolina State University Graduate Students (completed degree in bold): Jina Yu, Ph.D., Department of Food Science and Human Nutrition, Michigan State University Huan Zhang, Ph.D., Plant Pathology & Microbiology, Texas A&M University Huijuan Yan, Ph.D., Plant Pathology & Microbiology, Texas A&M University Chun-Chi Chen, Ph.D., Texas A&M, Dept. Electrical & Computer Engineering Hyun-Myung Woo, Ph.D., Texas A&M, Dept. Electrical & Computer Engineering Megan Sexton, Ph.D., Department of Plant Pathology, North Carolina State University Richard Gell, Ph.D., Department of Plant Pathology, North Carolina State University Weiran Li, Ph.D., Department of Botany and Plant Pathology, Purdue University Post Docs: Chen Chen, Michigan State University Chenxing Niu, Purdue University Nicole Mitchell, Michigan State University Mansuck Kim, Texas A&M University How have the results been disseminated to communities of interest?Oral/ Poster Presentations: Carbone, I. 2017. Identifying the players: dealing with diversity. Microbiome AgBioTechSummit. Raleigh, NC. Feb 21-23. Garcia, A., K.A. Wise, N.R. Anderson, J.D. Ravellette, S. Nelson, and G. Schuster. Field Inoculation Methods for Screening Corn Germplasm to Northern Corn Leaf Blight (Exserohilum turcicum) and Diplodia Leaf Streak (Stenocarpella macrospora). 28th Annual Texas Plant Protection Conference, December 6-7, 2016, Bryan, TX. Garcia, A., K.A. Wise, N.R. Anderson, J.D. Ravellette, S. Nelson, and G. Schuster. Field Inoculation Methods for Screening Corn Germplasm to Northern Corn Leaf Blight (Exserohilum turcicum) and Diplodia Leaf Streak (Stenocarpella macrospora). Pathways to the Doctorate Symposium, Texas A&M, Nov.4-5, 2016. Prairie View, TX Gell, R.M. and Carbone, I. 2017. Development of a Genetic Map for Aspergillus flavus. NCSU Genetics Program Retreat. Raleigh, NC. Aug 4. Gell, R.M. and Carbone, I. 2017. Sexual fertility in Aspergillus flavus: understanding genetic exchange and phenotypic inheritance through analysis of F1 progeny. The 29th Fungal Genetics Conference., Pacific Grove, CA. Mar 14-19. Gell, R.M. and Carbone, I. 2017. Sexual fertility in Aspergillus flavus: understanding genetic exchange and phenotypic inheritance through analysis of F1 progeny. The 14th International Aspergillus, Pacific Grove, CA. Mar 13-14. Gell, R. M. and Carbone, I. 2017. Sexual fertility in Aspergillus flavus: understanding genetic exchange and phenotypic inheritance through analysis of F1 progeny. The Mid-Atlantic States Mycology Conference (MASMC), Raleigh, NC. Apr 21-23. Isakeit, T., et al., 2016. Influence of corn GMO traits for insect control on aflatoxin and fumonisin contamination in Texas. Phytopathology 106 (Suppl. 4):4.46. Kim, M. and Shim, W.-B. 2017. Computational prediction of time-course subnetwork modules associated with histidine kinase activities in maize pathogen Fusarium verticillioides. American Phytopathological Society Annual Meeting. San Antonio, TX. Aug 5-9. Luis, J.M., et al., 2017. Morphological characterization, transcription profiling and metabolic expression during ascospore formation within fertilized sclerotia of Aspergillus flavus. The 29th Fungal Genetics Conference, Pacific Grove, CA. Mar 14-19. Luis, J.M., et al., 2017. Morphological, metabolic and transcriptional characterization of sexual fertilization in Aspergillus flavus. American Phytopathological Society Annual Meeting. San Antonio, TX. Aug 5-9. Mays, D., G.L. Schuster, M. Setamou, and S. Nelson Interaction between Bt and non-Bt corn hybrids and Afla-Guard to reduce aflatoxin contamination. Phytopathological Society Meeting, August 5-9, 2017, San Antonio, TX. Molo, M., et al., 2017. The population genomics underlying biological control of Aspergillus flavus and aflatoxin production. Plant Pathology PhD Symposium. Raleigh, NC. Apr 28. Molo, M et al., 2017. Population structure of Aspergillus flavus in soil and corn kernels from four different states. The 29th Fungal Genetics Conference. Pacific Grove, CA. Mar 14-19. Molo, M., et al., 2017. Population structure of Aspergillus flavus in soil and corn kernels from four different states. The 14th International Aspergillus Meeting. Pacific Grove, CA. Mar 13-14. Molo, M., et al., 2017. Population structure of Aspergillus flavus in soil and corn kernels from four different states. Mid-Atlantic States Mycology Conference (MASMC). Raleigh, NC. Apr 21-23. Moore, G.G., et al., 2017. A phylogeographic study of species from Aspergillus section Flavi. Mycological Society of America Annual Meeting. Athens, GA. Jul 16-19. Ojiambo, P.S. and Carbone, I. 2017. Female fertility and its role in selecting effective biocontrol strains of A. flavus. Opportunities for Reduction of Aflatoxin Contamination of Food. Cold Spring Harbor Laboratory, Lloyd Harbor, NY. Jul 9-12. Reese, B.N., et al., 2017. Corn Mycotoxins. National Association of County Agriculture Agents Annual Meeting and Professional Improvement Conference. Salt Lake City, UT. Jul 9-13. Wu, F. 2017. Opportunities for Reduction of Aflatoxin Contamination of Food. Cold Spring Harbor Laboratory, Lloyd Harbor, NY. Jul 9-12. Ridenour, J.B. et al., 2017. Casting a new die: Integrating novel biological insights and translational approaches to combat mycotoxin problems in maize. American Phytopathological Society Annual Meeting. San Antonio, TX. Aug 5-9. Salinas, Daniel , et al., 2017. Key Elements of Management for High Corn Yields within Eastern North Carolina. Pathways to the Doctorate Symposium, Texas A&M, Nov.4-5, 2016. Prairie View, TX. Schuster, G.L., et al., Influence of Bt maize (Zea mays (L.)) trait packages on aflatoxin contamination in south Texas maize production. Phytopathological Society Meeting, August 5-9, 2017, San Antonio, TX. Thomas, A., et al., Genetic structure, geography and host specialization within populations of Pseudoperonospora cubensis in the United States. American Phytopathological Society Annual Meeting. San Antonio, TX. Aug 5-9. Thomas, A., et al., 2017. Role of host types in shaping the genetic structure of Pseudoperonospora cubensis populations in the United States. The Oomycete Molecular Genetics Network Annual Meeting. Asilomar, CA. Mar 11-14. Wise, K.A. 2016. What I tell farmers about fungicides in field crops. Monsanto Lecture Series, Chesterfield, MO. Wise, K.A et al., Management of Aspergillus and Fusarium ear rots. Annual meeting of the American Phytopathological Society, Tampa Bay, FL. Aug 2016. Phytopathology 102:177. Wise, K.A., Isakeit, T., Heineger, R. and Woloshuk, C.P. 2016. Management of Aspergillus and Fusarium ear rots. Korean Society of Plant Pathology Annual Meeting, Pyeongchang, South Korea, October 2016. Woloshuk, C.P. 2017. Integrated management strategies for Aspergillus and Fusarium ear rots of corn. Agricultural Research Congressional Exhibition and Reception. Washington, DC. Apr 5. Woloshuk, C. P. Response to Fusarium verticillioides to maize kernel environment. Annual meeting of the Korean Society of Plant Pathology. Pyeongchang, Korea. October 2016. Wu, F. 2017. Understanding the risk from mycotoxins in animal-source foods. USAID Livestock Systems Innovation Laboratory Symposium: Nurturing Development. Gainesville, FL. Mar 30. Yan, H., et al., 2017. FRN1, a gene encoding putative GPCR protein, is critical for ear rot and fumonisin biosynthesis in maize pathogen Fusarium verticillioides. American Phytopathological Society Annual Meeting. San Antonio, TX. Aug 5-9. Yan, H., et al., 2017. Characterization of functional subnetwork modules associated with ear rot and fumonisin biosynthesis in maize pathogen Fusarium verticillioides. American Phytopathological Society Southern Division Meeting. College Station, TX. Feb 17-20. Yu, J., et al., 2017. The impact of Bt corn on aflatoxin occurrence. Annual meeting of the American Agricultural Economics Association. Chicago, IL. Jul 30-Aug 1. Zhang, H., et al., 2017. Characterization of a novel LysM/Chitin-binding protein FvLcp1 provides new insight into the regulation of ear rot virulence and fumonisin biosynthesis in Fusarium verticillioides. American Phytopathological Society Annual Meeting. San Antonio, TX. Aug 5-9. What do you plan to do during the next reporting period to accomplish the goals? Deployment of the Mac OS mobile app. (Theme 1) We will produce extension videos on ear rot identification, mycotoxin information, and grain handling protocols. The videos will be used during winter extension training activities and before harvest in the fall of 2018. (Theme 1) We have several extension bulletins that focus on the technologies developed. The extension bulletins will then be completed and incorporated into training activities. (Theme 1) We have collected hundreds of samples from field plots in North Carolina, Arkansas, Texas and Indiana. These samples are then processed by DNA extraction and sequencing for population analysis (Theme 2 Our research led to the discovery of a new protocol for identifying biocontrol strains within field populations. We anticipate completion of a patent application and marketing activities. (Theme 2) We will complete the analyze of the RNAi transgenic lines. (Theme 3) Research in Theme 3 has led to new patentable transgenic plants. We anticipate progress in the patent application and the release of several research manuscripts are being withheld until the patent is filed. (Theme 3) We anticipate that the computational network prediction modeling for Fusarium ear rot and Aspergillus ear rot will be completed. (Theme 3)

Impacts
What was accomplished under these goals? We determined that the cost of aflatoxin to US corn growers can vary from as low as $52 million in a "low-aflatoxin-risk" year to over $1 billion in a high-aflatoxin-risk year (Theme 4). In the area of BMP's, our most notable finding is that foliar fungicides are ineffective for mycotoxin control, and that farmers would be better served to employ biocontrol strains (Theme 1) or Bt corn (Theme 4). Building from this, the Theme 2 work has identified several new candidate biocontrol strains, which may be better suited to the states from which they were isolated than current commercially-available biocontrol products. We are also making considerable progress to understand the basic genetic characteristics that allow a biocontrol strain to remain dominant in a field for several years following one application. The novel computational method developed in Theme 3 has successfully identified candidate genes whose expression is associated with disease and mycotoxin production. We have successfully expressed interfering RNA's against several of these gene targets in corn cultivars and the pathogens. Our results show that interfering with the expression of these genes significantly lowers mycotoxin levels. Finally, we have communicated our findings and recommendations to our clientele through publications, workshops, exhibits, Internet websites and news releases.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Moore, G.G., Olarte, R.A., Horn, B.W., Elliott, J.L., Singh, R., ONeal, C.J. and Carbone, I. 2017. Global population structure and adaptive evolution of aflatoxin producing fungi. Ecology and Evolution DOI: 10.1002/ece3.3464.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Isakeit, T. 2016. Aspergillus Ear Rot and Aflatoxin. In: A Farmers Guide to Corn Diseases. K. Wise, D. Mueller, A. Sisson, D. Smith, C. Bradley, and A. Robertson, eds., APS Press, St. Paul, MN. pp.107-109.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Niu, C., Payne, G. A., Woloshuk, C. P. 2016. Involvement of FST1 from Fusarium verticillioides in virulence and transport of inositol. Molecular Plant Pathology DOI: 10.1111/mpp.12430.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: OKeeffe, K.R., Carbone, I., Jones, C.D. and Mitchell, C.E. 2017. Plastic potential: How the phenotypes and adaptations of pathogens are influenced by microbial interactions within plants. Current Opinion in Plant Biology 38: 78-83.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ridenour J. and Bluhm B. H. 2017. The novel fungal-specific gene FUG1 has a role in pathogenicity and fumonisin biosynthesis in Fusarium verticillioides. Molecular Plant Pathology 18:513-528.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Thomas, A., Carbone, I., Choe, K., Quesada-Ocampo, L.M. and Ojiambo, P.S. 2017. Resurgence of cucurbit downy mildew in the United States: Insights from comparative genomic analysis of Pseudoperonospora cubensis. Ecology and Evolution, DOI: 10.1002/ece3.3194.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wu, F. and Mitchell, N.J. 2016. How climate change and regulations can affect the economics of mycotoxins. World Mycotoxin Journal 9:653-663.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zaccaron, A. Z., Woloshuk, C. P., and Bluhm, B. H. 2017. Comparative genomics of maize ear rot pathogens reveals expansion of carbohydrate-active enzymes and secondary metabolism backbone genes in Stenocarpella maydis. Fungal Biology 11:966-983.


Progress 01/01/16 to 12/31/16

Outputs
Target Audience:Our project goals are 1) to provide corn growers with current best management practices for reducing ear rot diseases caused by the fungi Aspergillus flavus and Fusarium verticillioides and contamination with aflatoxins and fumonisins, respectively; and 2) to develop new management tools based on innovative genomic approaches. Our targeted audiences are corn producers, grain handlers, grain marketers, government officials, educators, and our scientific peers. Our communication methods include workshops, extension publications, websites, videos, peer reviewed publications, and oral/poster presentations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate Students: Ali Al-Rawi, PhD, Department of Plant Pathology University of Arkansas Angelyn Hilton, MS, Department of Plant Pathology and Microbiology, Texas A&M University Huan Zhang, PhD, Department of Plant Pathology and Microbiology, Texas A&M University Huijuan Yan, PhD, Department of Plant Pathology and Microbiology, Texas A&M University Jina Yu. PhD, Department of Agricultural, Food, and Resource Economics, Michigan State University John Ridenour, PhD, Department of Plant Pathology University of Arkansas Jonathon Smith, PhD, Department of Plant Pathology University of Arkansas Man S. Kim, PhD, Department of Electrical and Computer Engineering, Texas A&M University Megan Sexton, PhD, Department of Plant Pathology, North Carolina State University Richard Gell, PhD, Department of Plant Pathology, North Carolina State University Sandeep Sharma, PhD, Department of Plant Pathology, University of Arkansas Weiran Li, PhD, Department of Botany and Plant Pathology, Purdue University Post Docs: Dr. Angel Medina, Visiting Research Fellow (Cranfield University). North Carolina State University with Dr. Payne Dr. Braham Dhillon, Post-doctoral Associate. University of Arkansas with Dr. Bluhm Dr. Chenxing Niu, Post-doctoral Associate. Purdue University with Dr. Woloshuk Dr. Elizabeth Atungulu, Post-doctoral Associate. University of Arkansas with Dr. Bluhm Dr. Haozhen Nie, Post-doctoral Associate. Purdue University with Dr. Woloshuk Dr. Jimmy Man Kim, Post-doctoral Associate. Texas A&M University with Dr. Shim Dr. Karthi Keyan, Singh-Obama Postdoctoral Fellow. University of Arkansas with Dr. Bluhm Dr. Li Zhu, Visiting Scholar (China Research Council). University of Arkansas with Dr. Bluhm Dr. Nicole Mitchell, Post-doctoral Associate. Michigan State University with Dr. Wu Dr. Soumen Nandy, Post-doctoral Associate. University of Arkansas with Dr. Bluhm Dr. Yamunarani Ramegowda, Post-doctoral Associate. University of Arkansas with Dr. Bluhm Undergraduates: Alberto de la Garza, TAMUK intern at TAMUK (2015) Alvaro Garcia, TAMUK intern with Dr. Wise at Purdue (2015 & 2016) Cynthia Puente andJacobo Solis, TAMUK intern with Dr. Schuster at TAMUK (2016) Danny Salinas and Brance Crowell, TAMUK intern with Dr. Heiniger at NCSU (2016) Jacobo Solis, TAMUK intern with Dr. Wise at Purdue (2015 & 2016) Haley Higgins, University of Arkansas with Dr. Bluhm Tim Yeldell, University of Arkansas with Dr. Bluhm Trent LeMaster, TAMU with Dr. Shim Wayne Hawkins, University of Arkansas with Dr. Bluhm Wenting Liu, TAMU with Dr. Shim How have the results been disseminated to communities of interest? Field days and workshops: Woloshuk, Managing mold and mycotoxins in grain, Workshop, Clinton, IN County, Indiana, February 12, 2016, 119 Woloshuk, Managing mold and mycotoxins in grain, Weaver Popcorn Workshop, April 11, 2016, 30 Woloshuk, Mycotoxins and Post-Harvest Grain Handling, Post-Harvest Workshop, Purdue, DTC, August 25, 2016, 15 Woloshuk, Disease Issues in Corn: Ear Rots and Storage, Pinney Purdue Crop Diagnostic Training, September 7, 2016, 40 Wise, Corn disease update, Martin County program, November 18, 2015, 52 Wise, Ear rot management, Miami County program, December 3, 2015, 40 Wise, Corn disease management, Warrick County program, Boonville, IN, December 17 2015, 30 Wise, Corn disease management and ear rot concerns, Perry County program, Chrisney, IN, December 17, 2015, 40 Isakeit, Grain disease update, Upper Gulf Coast Feed Grain Conference, El Camp, TX, January 14, 2016, 90 Oral/ Poster Presentations: Bowman, J., Leslie, J. and Wu, F. 2015. How mycotoxins impact agriculture, nutrition and development. USAID AgriLinks, Washington, DC. May 20. Carbone, I. Sexton, M., Gell, R. M., White, J. B., Singh, R., Isakeit, T., Bluhm, B. H., Wise, K.A., Woloshuk, C.P., Horn, B. W. and Heiniger, R. 2016. Developing the next generation of biological control: genomic approaches for predicting most effective strains. American Phytopathological Society Annual Meeting. Tampa, FL. Jul 30 - Aug 3. Gell, R. M. and Carbone, I. 2016. Understanding sexual fertility in Aspergillus flavus through analysis of F1 progeny. American Phytopathological Society Annual Meeting. Tampa, FL. Jul 30-Aug 3. Hilton, A., Kim, M., Ortiz, C. Woloshuk, C.P., Yoon, B-J. and Shim, W-B. 2016. Computational and functional analyses of MAD-box transcription factor-mediated regulation of fumonisin biosynthesis in Fusarium verticillioides. American Phytopathological Society Annual Meeting, Tampa, FL. Jul 30-Aug 3. Isakeit, T., Murray, S.C. and Pekar, J. 2016 Influence of corn GMO traits for insect control on aflatoxin and fumonisin contamination in Texas. The American Phytopathological Society Meeting. Tampa, Florida. July 30- Aug 3. Kim, M., Yoon, B-J. and Shim, W-B. 2016. Systematic computational network-based analysis to predict subnetwork modules associated with pathogenicity and fumonisins in Fusarium verticillioides. American Phytopathological Society Annual Meeting, Tampa, FL. Jul 30-Aug 3. Kim, M., Yoon, B-J. and Shim, W-B. 2016. Systemic network-based computational identification of functional modules associated with pathogenicity and fumonisins in Fusarium verticillioides. 3rd Annual Texas A&M University ENG-LIFE Workshop: At the Interface of Engineering and Life Sciences, College Station, TX. Apr 29. Kim, M., Zhang, H., Yoon, B-J., Woloshuk, C. P. and Shim, W-B. Computational identification of functional network modules associated with Fusarium ear rot and fumonisin production. 2016 Corn Utilization and Technology Conference, St. Louis, MO. Jun 6-8. Mays, D., Schuster, G., Porter, P. and Setamou, M. 2015. Application timing of AF-36 and Afla-Guard in corn production to control mycotoxins. 69th Annual Meeting of the Subtropical Agriculture and Environments Society, Weslaco, TX. Feb 6. (Published in Subtrop. Plant Sci. Vol. 69). Mays, D., G. Schuster, P. Porter, M. Setamou, S.D. Nelson. 2016. Reducing mycotoxin levels in corn via lepidopteran insect control and application of atoxigenic fungi. American Phytopathological Society Annual Meeting, Tampa, FL. Jul 30-Aug 3. Mays, D., Schuster, G., Setamou, M. and Porter, P. 2015. Mycotoxin management in maize (Zea Mays (L.)) damaged by lepidopteran insect pests. 12th Annual Texas A&M System Student Research Symposium. Texas A&M University-Corpus Christi, TX. Oct 22-23. Mays, D., Schuster, G., Setamou, M., Porter, P., Wester, D., Tri, A. 2015. Mycotoxin management in maize (Zea mays (L.)) damaged by lepidopteran insect pests. American Phytopathological Society Annual Meeting, Pasadena, CA. Jul 31-Aug 5. Niu, C., Woloshuk, C. P. and Payne, G.A. 2015. Analysis of FST1 mutant of Fusarium verticillioides reveals global impact on transcriptome. American Phytopathological Society Annual Meeting, Pasadena, CA. Aug 3. Runa, F., Carbone, I., Horn, B. W., Bhatnagar, D. and Payne, G.A. 2015. Ploidy shift under high temperature and its effect on Aspergillus flavus ecology. American Phytopathological Society Annual Meeting. Pasadena, CA. Aug 1-5. Sexton, M., White, J.B., Isakeit, T. and Carbone, I. 2016. Temporal succession of an Aspergillus flavus biocontrol agent in commercial cornfields in Texas. American Phytopathological Society Annual Meeting. Tampa, FL. Jul 30-Aug 3. Shim, W-B. 2016. Systemic computational network-based analyses to predict functional subnetwork modules in maize - Fusarium interactions. 3rd Annual Texas A&M University ENG-LIFE Workshop: At the Interface of Engineering and Life Sciences, College Station, TX. Apr 29. Shim, W-B. 2016. Characterizing fumonisin B1 regulation in Fusarium verticillioides through computational subnetwork module analysis. Special Seminar. Dong A University, Pusan, South Korea. June 16. Shim, W-B. 2016. A new approach to identify gene targets for reducing fumonisin contamination. Corn Utilization and Technology Conference. St. Louis, MO. Jun 6-8. Shim, W-B. 2016. Computational identification of gene targets for reducing fumonisin contamination. Brain Korea 21 Seminar. Jinju, South Korea. Jun 17. Shim, W-B, Kim, M., Zhang, H., Yan, H., Yoon, B-J., Payne, G.A. and Woloshuk, C.P. 2016. Identifying new genetic targets for maize ear rot and mycotoxin control through computational subnetwork module analyses. American Phytopathological Society Annual Meeting, Tampa, FL; Jul 31-Aug 3. Woloshuk, C. P. 2016. Underlying mechanisms that regulate fumonisin biosynthesis in Fusarium verticillioides. North Carolina State University, Raleigh, NC. April 13. Wu, F. 2015. Food contaminants around the world: Impact of aflatoxin on global food trade. Kellogg Biological Station, Hickory Corners, MI. Sept 11. Wu, F. 2016. How food trade affects food safety: A case study of aflatoxin in maize and pistachios. Fate of the Earth Symposium, Michigan State University, East Lansing, MI. Apr 6-8. Yan, H. Zhang, H., Kim, M., Yoon, B-J. and Shim, W-B. 2016. Characterization of functional subnetwork modules associated with ear rot and fumonisin biosynthesis in maize pathogen Fusarium verticillioides. American Phytopathological Society Annual Meeting, Tampa, FL. Jul 30-Aug 3. Zaccaron, A., Ridenour, J.B., Smith, J.E., Woloshuk, C.P. and Bluhm, B.H. 2016. Draft genome sequence of Stenocarpella maydis as a resource for molecular genetics and functional genomics. Oral presentation by A. Zaccaron, American Phytopathological Society/Southern Division, Annual Meeting, Balm, FL. Feb 20-22. Zhang, H., Kim, M., Yan, H., Yoon, B-J. and Shim, W-B. 2016. Enhancing our understanding of Fusarium maize stalk rot pathogenesis through gene association subnetwork module analyses. The American Phytopathological Society Meeting. Tampa, FL. Jul 30-Aug 3. What do you plan to do during the next reporting period to accomplish the goals?Under goal 1, an experiment will be conducted in Texas to compare the impact of strip vs. uniform application of an atoxigenic A. flavus strain on aflatoxin and fumonisin. Another experiment will compare aflatoxin and fumonisin levels in two commercial fields previously treated with atoxigenic strains, following treatment during the current season with a 5 lb./A rate, in comparison with a 10 lb./A rate or no treatment. Research results on improved mycotoxin management will be delivered to stakeholders through various formats in 2017, including presentations at Extension meetings, and via our cornmycotoxins.com website. Also we will be establishing booths at farm trade shows to increase the visibility of the project results with farmers and increase awareness of the project outputs. Under goal 2, We plan to use genotyping by sequencing (GBS) methods to genotype 1,152 Fusarium verticilliodes isolates from corn kernels sampled over three years (2013-2016).We will patent the new Aspergillus flavus candidate biocontrol strains and the process used to select them. Also we will estimate recombination rate and extrapolate these results to field data to predict which strains and propagules (sclerotia or conidia) will be most effective in spreading favorable genetic determinants in the population. Under goal 3, We will complete the validation of ten F. verticillioides and five A. flavus functional subnetwork modules predicted to exhibit significant influence on ear rot pathogenesis and mycotoxin biosynthesis. We will advance lines of transgenic corn targeting fungal genes that have provided promising initial results regarding suppression of mycotoxin biosynthesis during pathogenesis. Also we will evaluate transgenic corn lines targeting hxk, aflA and aflS. Under goal 4, we will evaluate the benefit-cost and cost-effectiveness of each of the different technologies and strategies developed in Themes 1, 2, and 3 of our project. Also we will evaluate the current regulatory regime in the United States for the mycotoxin control strategies we have developed.

Impacts
What was accomplished under these goals? Under goal 1, we have completed experiments to determine BMPs for mycotoxin management. Over the three years, we determined that consistency is variable in application timing for aflatoxin and fumonisin control, but early applications (V5-V7) may have some impact on reducing mycotoxins in certain years. This information allows farmers flexibility in their atoxigenic application timing, but consistency from year to year is influenced by aflatoxin levels. Hybrids with multiple Bt traits (Viptera) may be more beneficial in reducing aflatoxin and fumonisin in Texas than hybrids with the Cry1Ab trait alone. In 2016, five new Extension publications were developed to provide updated information on ear rots, mycotoxins, grain testing and storage to stakeholders. . Links to the publications were added to our farmer-targeted website (www.cornmycotoxins.com), which receives frequent traffic from the U.S. and around the world. The website has been advertised to over 1,000 participants at extension field days and workshops, and through newsletters. Under goal 2, our research results provide a better picture of what may happen when farmers apply a biocontrol agent to their corn field. Although the Afla-Guard® strain itself may not prevail through successive growing seasons, our results suggest that the strain may sexually recombine with native strains. Also results provide evidence that supports a genetic approach for selecting biocontrol strains. If male conidia of a highly fertile sclerotial parent are used as a biocontrol, we would expect it to spread fertility genes into the native population. Conversely, low fertility sclerotial females can shift the balance towards sterility. The extent to which these factors may contribute to longevity of biological control is currently under investigation. Last year we deployed several of these new biocontrol candidate strains in each state. So far, results from NC indicate significant differences among the strains and the untreated control for aflatoxin contamination. Under goal 3, our research results successfully demonstrated that a HIGS-based (RNAi) approach could work to control toxin production by mycotoxigenic fungi in corn. Also our computational prediction-pipeline and experimental validation has identified new target gene for our RNAi approach, which would be overlooked by other screening methods. Under goal 4, our research evaluated aflatoxin-related economic loss to the US corn industry and potential future scenarios with a warmer climate and possibly more of the Midwestern corn-planting states affected by aflatoxin contamination.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Horn, B.W., Gell, R.M., Singh, R., Sorensen, R. B. and Carbone, I. 2016. Sexual reproduction in Aspergillus flavus sclerotia: acquisition of novel alleles from soil populations and uniparental mitochondrial inheritance. PloS One 11(1): e0146169. PMID: 26731416.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mitchell, N. J., Bowers, E., Hurburgh, C. and Wu, F. 2016. Potential economic losses to the US corn industry from mycotoxin contamination. Food Additives and Contaminants 33:540-50.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Niu, C., Payne, G. A. and Woloshuk, C. P. 2016. Structural and functional analysis of FST1 in Fusarium verticillioides. Molecular Plant Pathology DOI: 10.1111/mpp.12430
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Payne, G.A. 2016. Mycotoxins and Product Safety. In: Stalker, H.T., Wilson, R.F. (Eds.), Peanuts: Genetics, Processing, and Utilization. Academic Press and AOCS Press, pp. 347361. ISBN: 9781630670382.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ridenour, J. and Bluhm, B. H. 2016. The novel fungal-specific gene FUG1 has a role in pathogenicity and fumonisin biosynthesis in Fusarium verticillioides. Molecular Plant Pathology DOI: 10.1111/mpp.12414.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Ridenour, J. and Bluhm, B. H. 2016. The HAP complex of Fusarium verticillioides regulates fumonisin biosynthesis and kernel pathogenesis. Journal of Food Protection (in press).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Runa, F., Carbone, I., Bhatnagar, D. and Payne, G. A. 2015. Nuclear heterogeneity in conidial populations of Aspergillus flavus. Fungal Genetics and Biology 84: 6272.
  • Type: Theses/Dissertations Status: Other Year Published: 2016 Citation: Chenxing Niu, (2016) Functional and structural analysis of FST1 in Fusarium verticillioides. PhD, Purdue University.
  • Type: Theses/Dissertations Status: Other Year Published: 2016 Citation: Tyler Mays (2016) Mycotoxin management in maize (Zea mays): damaged by lepidopteran pests. MSc, Texas A&M University, Kingsville
  • Type: Other Status: Published Year Published: 2016 Citation: Isakeit, T., Allen, T., Chilvers, M., Faske, T., Freije, A., Mueller, D., Price, T., Smith, D., Tenuta, A., Wise, K., and Woloshuk, C. 2016. Corn Disease Management: Using atoxigenics to manage aflatoxin. Crop Protection Network. CPN 1105. http://cropprotectionnetwork.org/corn-diseases/using-atoxigenics/
  • Type: Other Status: Published Year Published: 2016 Citation: Faske, T., Allen, T., Chilvers, M., Freije, A., Isakeit, T., Mueller, D., Price, T., Smith D., Tenuta, A., Wise, K., and Woloshuk, C. 2016. Corn Disease Management: Storing mycotoxin-affected grain. Crop Protection Network. CPN 1104. http://cropprotectionnetwork.org/corn-diseases/storing-mycotoxin-affected-grain/
  • Type: Other Status: Published Year Published: 2016 Citation: Woloshuk, C., Allen, T., Chilvers, M., Faske, T., Freije, A., Isakeit, T., Mueller, D., Price, T., Tenuta, A., and Wise, K. 2016. Corn Disease Management: Grain sampling and mycotoxin testing. Crop Protection Network. CPN 1103. http://cropprotectionnetwork.org/corn-diseases/grain-sampling-mycotoxin-testing/
  • Type: Other Status: Published Year Published: 2016 Citation: Allen, T., Chilvers, M., Faske, T., Freije, A., Isakeit, T., Mueller, D., Price, T., Smith, D., Tenuta, A., Wise, K., and Woloshuk, C. 2016. Corn Disease Management: Mycotoxin FAQs. Crop Protection Network. CPN 1102. http://cropprotectionnetwork.org/corn-diseases/mycotoxin-faqs/
  • Type: Other Status: Published Year Published: 2016 Citation: Wise, K., Allen, T., Chilvers, M., Faske, T., Freije, A., Isakeit, T., Mueller, D., Price, T., Sisson, A., Smith, D., Tenuta, A., and Woloshuk, C. Corn Disease Management: Ear rots. Crop Protection Network. CPN 1101. http://cropprotectionnetwork.org/corn-diseases/ear-rots/
  • Type: Theses/Dissertations Status: Other Year Published: 2016 Citation: Angelyn Hilton (2016) Investigation of Fusarium stalk and kernel rot diseases in field crops. MSc, Texas A&M University
  • Type: Theses/Dissertations Status: Other Year Published: 2016 Citation: Man S. Kim. (2016) Computational identification of functional modules and hub genes involved in pathogenicity-associated or defense response on Fusarium verticillioidesmaize interactions. PhD, Texas A&M University


Progress 01/01/15 to 12/31/15

Outputs
Target Audience:Our project goals are 1) to provide corn growers with current best management practices for reducing ear rot fungi diseases caused byAspergillus flavusandFusarium verticillioidesand contamination with aflatoxins and fumonisins, respectively; and 2) to develop new management tools based on innovative genomic approaches. Although a complete report on the 2015 outcomes on field experiments that are still ongoing need to be completed, we have made progress towards our goals. First, we have now completed the assessment of the impact of fungicides on Aspergillus and Fusarium ear rots, and our assessment of the applications of the biocontrol products AflaGuard and AF-36. The results indicate fungicides are not able to consistently reduce mycotoxins, but do not negatively affect atoxigenic biocontrol strains. Furthermore, timing of atoxigenic biocontrol applications can vary and farmers have more flexibility in application timing than originally thought. To communicate these updated best management practices to our clientele, we have launched a user-friendly Internet site (cornmycotoxins.com). We have advertised the website through a variety of means, including farmer meetings, news releases, and radio interviews. We have consistently had about 100 new visitors to the site each week. We have also begun the development of a phone app that mirrors the Internet site. A beta version will be demonstrated and discussed at the annual meeting in November. Progress also has been made in our effort to identify new biocontrol strains ofA. flavus. Based on 2013 and 2014 population results, we identified state-specific strains from NC, IN, TX, and AR with the desired genotype characteristics. These strains are being tested in the 2015 field experiments. We have developed network algorithms that allow us to identify potential gene targets for inhibiting fumonisin and aflatoxin. Whole genome expression data were analyzed and several genes were identified as candidates. These are being expressed as RNAi in transgenic corn. Finally, we have established the baseline values for economic losses caused by aflatoxin and fumonisin contamination in the US. We found that both regional and year-to-year climatic condition greatly influence the losses. Changes/Problems:We have added Micheal Langemeier, Agricultural Economics at Purdue University to our research team. Dr. Langemeier will assist us in developing economic calculators for a smart phone App. What opportunities for training and professional development has the project provided?Graduate Students: Mansuck Kim. PhD student, Department of Electrical and Computer Engineering, Texas A&M University, College Station (2014 - Present) Richard Gell. PhD student, Department of Plant Pathology, Genetics Program, North Carolina State University (2014 - Present) Mary Lewis. MSc student, Department of Plant Pathology, North Carolina State University (2012 - 2014) Weiran Li. PhD student, Department of Botany and Plant Pathology, Purdue University (2014 - Present) Dennis Mays. MSc student, Texas A&M University, Kingsville (2014 - Present) Megan Meyers. MSc student, Department of Plant Pathology, North Carolina State University (2013 - 2014) Trent Monacell. PhD student, Bioinformatics & Plant Pathology; North Carolina State University (2008 - 2014) Rodrigo Olarte. PhD student, Department of Plant Pathology, North Carolina State University (2008 - 2014) Farhana Runa. PhD student, Department of Plant Pathology, North Carolina State University (2010 - 2014) Megan Sexton. PhD student, Departments of Plant Pathology and Crop Science, North Carolina State University (2013 - Present) Jina Yu. PhD student, Department of Agricultural, Food, and Resource Economics Michigan State University (2015 - Present) Huan Zhang. Ph.D. student, Department of Plant Pathology and Microbiology, Texas A&M University, College Station (2014 - Present) Jonathon Smith, PhD student, Department of Plant Pathology, University of Arkansas (2010 - present) John Ridenour, MS student, Department of Plant Pathology, University of Arkansas (2010 - 2014) Ali Al-Rawi, PhD student, Department of Plant Pathology, University of Arkansas (2015 - present) Post Docs: Dr. Nicole Mitchell mentored by Dr. Wu, Department of Agricultural, Food, and Resource Economics Michigan State University (2014-Present) Dr. Haozhen Nie mentored by Dr. Woloshuk, Department of Botany and Plant Pathology, Purdue University (2015) Dr. Yamunarani Ramegowda, mentored by Dr. Bluhm, Department of Plant Pathology, University of Arkansas (2012 - present) Dr. Marikunte Y. Sreenivasa, mentored by Dr. Bluhm, Department of Plant Pathology, University of Arkansas (2013 - 2014) Dr. Karthi Keyan, mentored by Dr. Bluhm, Department of Plant Pathology, University of Arkansas (2014 - present) Dr. Elizabeth Atungulu, mentored by Dr. Bluhm, Department of Plant Pathology, University of Arkansas (2013 - present) Undergraduates Dale Herrington, TAMUK intern with Dr. Heiniger, NCSU (2014) Kameron Koepp, TAMUK intern with Dr. Heiniger, NCSU (2014) Victor Gutierrez, TAMUK intern at TAMUK (2014) Deanna Cano, TAMUK intern at TAMUK (2014) Alberto de la Garza, TAMUK intern at TAMUK (2015) Cesar Canales, TAMUK intern at TAMUK (2015) Jacobo Solis, TAMUK intern with Dr. Wise at Purdue (2015) Alvaro Garcia, TAMUK intern with Dr. Wise at Purdue (2015) Steven Wrobliski, TAMUK student worker at TAMUK, (2015) Wayne Hawkins, UARK student and intern with Dr. Bluhm at Arkansas (2014-2015) Micah Velasquez, UARK student intern with Dr. Bluhm at Arkansas (2014 -2015) Tim Yeldell, UARK student intern with Dr. Bluhm at Arkansas (2014 - 2015) Annai Gonzalez, Undergraduate research assistant with Dr. Shim at TAMUCS (2014 -2015) Jose Macias. Undergraduate Research Scholar with Dr. Shim at TAMUCS (2015) How have the results been disseminated to communities of interest?Field days and workshops. Woloshuk, Managing molds and mycotoxins in grain, Post Harvest Workshop, Purdue University, August 21, 2014 Woloshuk, Stored grain molds, mycotoxins and post-harvest grain-handling, Southwest Indiana Crop Seminar, February 2, 2015 Woloshuk, Mycotoxins and post-harvest grain handling, Delaware County Field Crops Program, December 12, 2014 Isakeit, Aflatoxin control , 2015 Bell County Crops Conference, Belton, TX, January 20, 2015 Isakeit, Disease concerns in cotton, corn & sorghum. , Multi-county Winter Ag Conference, Stockdale, TX, January 28, 2015 Isakeit, Grain disease update , Upper Gulf Coast Feed Grain & Cotton Conference, Wharton, TX, January 29, 2015 Isakeit, Afla-Guard and AF 36 Update , Black Income Growth, Waco, TX, February 3, 2015 Isakeit, Field crop diseases , Fort Bend County Row Crops Tour, Rosenberg, TX, June 16, 2015 Isakeit, Corn and sorghum diseases , B-H Genetics 2015 Annual Field Day, Ganado, TX, June 17, 2015 Wise, Corn disease update: focus on ear rots, Kentuckiana Crop Conference, French Lick, IN, December 2, 2015 Wise, Certified Crop Advisor preparatory course on ear rot management, Diagnostic Training Center Program, West Lafayette, IN, January 13, 2015 Wise, Corn disease management, Shelby County Program, Shelbyville, IN, February 16, 2015 Wise, Corn disease management and ear rot concerns, Weaver popcorn training, West Lafayette, IN, March 6, 2015 Heiniger, Managing mycotoxins in corn in North Carolina, Southeastern Extension Agent Training, December 9, 2014 Heiniger, Managing mycotoxins with non-toxigenic strains, Bertie, Hertford, and Martin County Corn Meetings, January 29, 2015 Heiniger, Managing mycotoxins with non-toxigenic Strains, Northhampton County Meeting, February 17, 2015 Heiniger, Managing mycotoxins with non-toxigenic strains, Wayne and Greene County Corn Meetings, February 18, 2015 Heiniger, Managing mycotoxins with non-toxigenic strains, Rockingham County Corn Meeting, March 6, 2015 Heiniger, Managing mycotoxins with non-toxigenic strains, Robeson County Corn Meeting, March 11, 2015 Wu, Breaking the mold, US Agency for International Development (USAID) webinar, May 20, 2015 Wu, Reducing mycotoxins in diets worldwide: the potential role of transgenic Bt maize, Plant Biotechnology for Health & Sustainability, Michigan State University, October 31, 2014 Wu, A tangled web: Social network models of food toxin standards worldwide, & impacts on trade & health, Center for Integrative Toxicology, Michigan State University, October 31, 2014 What do you plan to do during the next reporting period to accomplish the goals?In the fourth year of this five-year research project, we will continue our effort toward finding and evaluating the next-generation biocontrol strains that are regionally adapted and more sustainable. We will test the efficacy of transgenic corn expressing RNAi technology that disrupts mycotoxin production. We will also continue to identify new molecular targets for disrupting mycotoxin production and validate those gene targets already identified. Finally, we will measure economic benefits and risks of these technologies to growers and end users. Our goal is to provide corn producers with new tools and access to up-to-date information about the best management strategies for reducing mycotoxin contamination. Material that we have generated will be communicated to clientele through the extension Internet site, field demonstrations, workshops, and videos. We will also launch an App that producers can access with their smart phone. Surveys of clientele will continue to help us measure their needs and the impact of our research.

Impacts
What was accomplished under these goals? Aflatoxins pose a significant health hazard and limit the marketability of US corn. Our research has estimated that in the southern US, where aflatoxin contamination in corn is a chronic occurrence, as much as $200 million are lost each year. We also estimate that during years when drought and high temperature affect the Midwest Corn Belt, losses can reach $1 billion. Corn producers need up-to-date information about the best management strategies for reducing mycotoxin contamination. We have measured that about 100 new viewers per week visit our Extension (grower-targeted) website, which indicates that our project is addressing this need. Our field research has shown - and we are communicating to growers - that recommendations need to be regionally specific, which include the recommendation that biocontrols can be applied from V7-V10 in Texas and North Carolina, and it is not economical to apply biocontrol in Indiana. We also recommend that fungicides not be applied to reduce plant stress or for ear rot/mycotoxin issues, although their application for foliar control of other pathogens will not affect biocontrol strains. Biocontrol is one of the important management tools for reducing contamination, especially in the chronic aflatoxin regions; at present, there are two commercially available products. Through our population genetics research, we have rationally selected new strains that are regionally specific for Texas, North Carolina, Arkansas, and Indiana, which have the inherent characteristics for superior biocontrol. We are testing the efficacy of these strains. Genetic modification (GMO) of corn is also an important management approach. We have discovered a network of fungal genes that appear to be viable targets for suppression by RNAi in GM corn. Preliminary data indicate that RNAi expression does, in fact, have significant promise for reducing aflatoxin contamination in corn.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Xiaomei Shu, David P. Livingston III, Robert G. Franks, Rebecca S. Boston, Charles P. Woloshuk, and Gary A. Payne. 2015. Tissue-specific gene expression in maize seeds during colonization by Aspergillus flavus and Fusarium verticillioides. Mol. Plant Path. DOI: 10.1111/mpp.12224
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kim, M., Zhang, H., Woloshuk, C., Shim, W.-B. and B.-J. Yoon. 2015.Computational identification of genetic subnetwork modules associated with maize defense response to Fusarium verticillioides. BMC Bioinformatics. 16:S12 doi:10.1186/1471-2105-16-S13-S12
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Miller, J. D., Schaafsma, A. W., Bhatnagar, D., Bondy, G., Carbone, I., Harris, L., Harrison, G., Munkvold, G., Oswald, I., Pestka, J., Sharpe, L., Sumarah, M., Tittlemier, S. and T. Zhou. 2014. Mycotoxins that affect the North American Agri-Food sector: state of the art and directions for the future. World Mycotoxin Journal 7 (1): 63-82.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Ridenour, J.B., Smith, J.E., Hirsch, R.L., Horevaj, P., Kim, H., Sharma, S., and B.H. Bluhm. 2014. UBL1 of Fusarium verticillioides encodes a putative E3 ubiquitin ligase involved in growth, conidiation, virulence, amylolysis and secondary metabolism. Environmental Microbiology 16: 2004-22.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Wu, F. 2015. Global impacts of aflatoxin in maize: Trade and human health. World Mycotoxin Journal 8:137-142.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Carbone, I. Sex, fertility and biocontrol in Aspergillus. 2015. Program in Genetics Seminar Series, North Carolina State University, Apr 20.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Dupnik, M., C.P. Woloshuk, G.L. Schuster and S.D. Nelson. 2014. Assessment of Aspergillus flavus and aflatoxin production under low oxygen conditions. Undergraduate Poster Competition, Subtropical Agriculture & Environments, Weslaco, TX. Feb 21.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Dupnik, M., C.P. Woloshuk, G.L. Schuster and S.D. Nelson. 2014. Assessment of Aspergillus flavus and aflatoxin production under low oxygen conditions. Undergraduate Poster Competition. Annual Meeting of the Southern Branch of the American Society of Agronomy, Dallas, TX. Feb 2-4.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Gell, R.M. and I. Carbone. 2015. Genetic determinants of sexual fertility in Aspergillus flavus. The 39th Annual Genetics Graduate Student Symposium, Raleigh, NC. Feb 13.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Monacell, J.T. and I. Carbone. 2014. Mobyle SNAP Workbench: A web-based analysis portal for population genetics and evolutionary genomics. Bioinformatics 30:1488-1490. DOI: 10.1093/bioinformatics/btu055 PMID: 24489366
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Niu, C., G.A. Payne and C.P. Woloshuk. 2015. Transcriptome changes in Fusarium verticillioides caused by mutation in the transporter-like gene FST1. BMC Microbiology. 15:90. DOI:10.1186/s12866-015-0427-3
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Olarte, R. A., Horn, B.W., Singh, R. and I. Carbone. 2015. Sexual recombination in Aspergillus tubingensis. Mycologia 107:307-312.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Olarte, R. A., Worthington, C.J., Horn, B.W., Moore, G.G., Singh, R., Monacell, J.T., Dorner, J.T., Stone, E.A., Xie, D-Y. and I. Carbone. 2015. Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus. Molecular Ecology 24:1889-1909.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Ridenour, J.B. and B.H. Bluhm. 2014. The HAP complex in Fusarium verticillioides is a key regulator of growth, morphogenesis, secondary metabolism, and pathogenesis. Fungal Genetics and Biology 69:52-64.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Gell, R.M., B.W. Horn and I. Carbone. 2015. Understanding the role of sexual fertility in selection of aflatoxin biocontrol strains. The Gordon Research Conference (GRC) on Mycotoxins and Phycotoxins. The Biology, Chemistry and Ecology of Naturally Occurring Fungal and Algal Toxins with Public Health and Economic Impacts. Stonehill College, Easton, MA; June 14-19.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Kim, M., H. Zhang, C. Woloshuk, W.-B. Shim and B.-J. Yoon. 2015. Computational identification of genetic subnetwork modules associated with maize defense response to Fusarium verticillioides. Midsouth Computational Biology and Bioinformatics Society Annual Conference, Little Rock, AR. March 13-14.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Lewis, M.H., I. Carbone, G.A. Payne, K.L. Bowen, A. Hagan, R. Kemerait, R. Heiniger and P.S. Ojiambo. 2014. Relationship between efficacy of biocontrol of aflatoxin in maize and genetic structure of native Aspergillus flavus. The APS-CPS Joint Meeting. Minneapolis, Minnesota. Aug 9-13.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Lewis, M. H., I. Carbone, G.A. Payne and P.S. Ojiambo. 2014. Implication of genetic structure of Aspergillus section Flavi in soil on efficacy of biocontrol of aflatoxin in corn. NCSU Graduate Student Research Symposium, McKimmon Center, Raleigh, NC. Mar 26.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Gell, R.M., B.W. Horn and I. Carbone. 2015. Understanding the role of sexual fertility in selection of aflatoxin biocontrol strains. The Gordon Research Seminar (GRS) on Mycotoxins and Phycotoxins. Harmful Biotoxins: Their Chemistry, Ecological Role and Associated Risks. Stonehill College, Easton, MA; June 13-14.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Herrington, D., G. Schuster, A. Lewis and P. Porter. 2014. Mycotoxin levels associated with fall armyworm (Spodoptera frugiperda) and corn earworm (Helicoverpa zea) infested corn. Undergraduate Poster Competition, Subtropical Agriculture & Environments, Weslaco, TX. Feb 21.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Herrington, D., G. Schuster, A. Lewis, and P. Porter. 2014. Mycotoxin levels associated with fall armyworm (Spodoptera frugiperda) and corn earworm (Helicoverpa zea) infested corn. Undergraduate Poster Competition (Won 3rd place). Annual Meeting of the Southern Branch of the American Society of Agronomy, Dallas, TX. Feb 2-4.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Isakeit, T. 2014. Evaluation of atoxigenic strains of Aspergillus flavus for reduction of aflatoxin contamination of maize on commercial farms in Texas, USA. 36th Mycotoxin Workshop of the Society for Mycotoxin Research, G�ttingen, Germany, June 18, 2014
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Kim, M., H. Zhang, C. Woloshuk, W.-B. Shim and B.-J. Yoon. Computational identification of functional network modules associated with the pathogenicity of Fusarium verticillioides. International Workshop on Computational Network Biology: Modeling, Analysis, and Control, Newport Beach, CA; September 20, 2014.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Kim, M., H. Zhang, C. Woloshuk, W.-B. Shim and B.-J. Yoon. 2015. Computational identification of key functional genes associated with Fusarium verticillioides pathogenicity. The 6th ACM Conference on Bioinformatics, Computational Biology, and Health Informatics. Atlanta, GA. Sept 9-12.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Mays, D., G. Schuster, M. S�tamou, P. Porter, D. Wester, A. Tri. 2015. Mycotoxin Management in Maize (Zea mays (L.)) Damaged by Lepidopteran Insects Pest. Annual Meeting of the American Phytopathological Society, Pasadena, CA. August 1-5.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Medina-Vaya, A., A. Rodr�guez, G. Payne, D. Bhatnagar, and N. Magan. 2015. Aspergillus flavus and climate change: Effects of environmental changes on growth, gene expression, and aflatoxin production in vitro and in maize. Gordon Research Conference on Mycotoxins and Phycotoxins. Stonehill College, Easton, MA. June 14-19.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Musungu, B., G. OBrian, R. Brown, D. Bhatnagar, G. Payne, A. Geisler and A. Fakhoury. 2015. Systematic and Temporal Analysis of Aspergillus flavus and Zea mays. Gordon Research Conference on Mycotoxins and Phycotoxins. Stonehill College, Easton, MA. June 14-19.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Musungu, B., R.G. OBrian, R Brown, D. Bhatnagar, G. Payne, M. Geisler and A.M. Fakhoury. 2014. Gene regulatory networks for resistance in maize to ear rots and to aflatoxin accumulation. Annual Meeting of the American Phytopathological Society, Minneapolis MN. August 10-14.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Payne, G.A. 2014. Genomic approaches to characterize regulator circuits in Aspergillus flavus controlling aflatoxin biosynthesis. Annual Meeting of the American Phytopathological Society, Minneapolis MN. August 10-14.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Sexton, M., R. Singh, J.B. White, T. Isakeit, B.H. Bluhm, K.A. Wise, C.P. Woloshuk, B.W. Horn, R. Heiniger and I. Carbone. 2015. A population genomics approach to understanding biological control in Aspergillus flavus. The Gordon Research Conference (GRC) on Mycotoxins and Phycotoxins. The Biology, Chemistry and Ecology of Naturally Occurring Fungal and Algal Toxins with Public Health and Economic Impacts. Stonehill College, Easton, MA; June 14-19.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Sexton, M., R. Singh, J.B. White, T. Isakeit, B.H. Bluhm, K.A. Wise, C.P. Woloshuk, B.W. Horn, R. Heiniger and I. Carbone. 2015. A population genomics approach to understanding biological control in Aspergillus flavus. The Gordon Research Seminar (GRS) on Mycotoxins and Phycotoxins. Harmful Biotoxins: Their Chemistry, Ecological Role and Associated Risks. Stonehill College, Easton, MA; June 13-14.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Shim, W.-B. 2014. Impact of extreme climate on mycotoxin and food security. 3rd Korea-Japan Joint Plant Pathology Meeting, Pusan, South Korea. October 23.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Shim, W.-B. 2015. Association network biology to discover regulatory logic underpinning maize-fungal interaction, Brain Korea 21 Program. Gyeongsang National University, Jinju, South Korea.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Niu, C., G.A. Payne and C.P. Woloshuk. 2015. Analysis of FST1 mutant of Fusarium verticillioides reveals global impact on transcriptome. Annual Meeting of the American Phytopathological Society, Pasadena, CA. August 1-5.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Ridenour, J., and B.H. Bluhm. 2014. Ubl1 links the N-end rule pathway to pathogenesis and extracellular sensing in Fusarium verticillioides. Invited presentation by J. Ridenour. Mycological Society of America, Annual Meeting, East Lansing, MI; June 8  12.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Shim, W.-B. 2015. Association network analysis to discover regulatory logic in maize-Fusarium interactions. Fujian Agricultural and Forestry University  Texas A&M University Symposium on Fungal Biology, Fuzhou, PR China.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Zhang, H., M. Kim, C.P. Woloshuk, B.J. Yoon and W.-B. Shim. 2015. Computational identification of functional network modules associated with fumonisin biosynthesis in maize pathogen Fusarium verticillioides. 28th Fungal Genetics Conference, Pacific Grove, CA. March 17-22.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Lewis, M. 2014. Genetic structure of soil populations of Aspergillus section Flavi and efficacy of biocontrol of aflatoxin contamination in corn. (under the direction of Dr. Ignazio Carbone and Dr. Peter S. Ojiambo). MSc Thesis, North Carolina State University.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Monacell, J. T. 2014. Identification of heterokaryon incompatibility genes in Aspergillus using array comparative genome hybridization and whole genome sequencing. (under the direction of Dr. Ignazio Carbone). PhD Thesis, North Carolina State University.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Olarte, Rodrigo, A. 2014. Population dynamics of intra- and inter-specific crosses and the effect of biocontrol on natural populations of Aspergillus species. (under the direction of Dr. Ignazio Carbone). PhD Thesis, North Carolina State University.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Ridenour, John. 2014. Characterization of a Novel Fungal-Specific Gene, FUG1, in Fusarium verticillioides. (under the direction of Dr. Burt Bluhm). MSc Thesis, University of Arkansas.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Runa, Farhana. 2014. Ploidy and nuclear condition in Aspergillus flavus and their effect on its ecology. (under the direction of Dr. Gary A. Payne and Dr. Ignazio Carbone). PhD Thesis, North Carolina State University.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Shu, Xaiomei. 2014. Pathogenesis and host response during infection of maize kernels by Aspergillus flavus and Fusarium verticillioides. (under the direction of Dr. Gary A. Payne). PhD Thesis, North Carolina State University.


Progress 01/01/14 to 12/31/14

Outputs
Target Audience: Over the past year we conducted field and greenhouse research in Indiana, Arkansas, Texas, and North Carolina. The results have been communicated to the scientific community through several professional meetings, journal, publications and grower meetings. We also continued our effort to explain to growers the current knowledge about aflatoxin and biocontrol strategies, and to explain our effort to develop and improve management tools for reducing the risks of mycotoxin contamination. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Dr. Nicole Mitchell is a postdoctoral research fellow mentored by Dr. Felicia Wu. Dr. Mitchell received training in assessing economic impacts of food contaminants for the purposes of this project. She has learned mathematical modeling and basic benefit-cost analyses, and has aided in data collection for aflatoxin and fumonisin contamination levels in various states. Mansuck Kim is a Ph.D. student, Department of Electrical and Computer Engineering, Texas A&M University, College Station, TX. Mr. Kim contributed to the publication submitted to IEEE/ACM Transactions on Computational Biology and Bioinformatics and will be the presenter of a research poster at the International workshop on computational network biology: Modeling, Analysis, and Control (Newport Beach, CA) Huan Zhang is a Ph.D. student, Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX. Ms. Zhang was primarily responsible for biological sample preparations for RNA-Seq and contributed to the publication submitted to IEEE/ACM Transactions on Computational Biology and Bioinformatics and the research abstract for the international workshop on computational network biology: Modeling, Analysis, and Control (Newport Beach, CA) Weiran Li is a PhD graduate student in the Department of Botany and Plant Pathology at Purdue University. Weiran’s research is focused on the project Objective 3.1 Establish biomarkers in corn that predict predisposition to mycotoxin contamination. Weiran’s academic advisor is Dr. Charles Woloshuk. Megan Meyers is a MSc student in Plant Pathology at North Carolina State University under the supervision of Drs. Carbone and Heiniger. Her research focuses on developing improved management strategies for reducing mycotoxins in corn (Theme 1). Megan Sexton is a PhD student co-majoring in Plant Pathology and Crop Science at North Carolina State University. Megan's research is focused on developing new biocontrol methods for mycotoxins (aflatoxin and fumonisin) in corn (Theme 2). Megan is co-advised by Drs. Carbone and Heiniger. Rakhi Singh is a Research Technician and Lab Manager. She was instrumental in developing the ddRADSeq protocol and testing/debugging analysis tools for analyzing NGS data. Dennis Mays is a MSc student at TAMUK working on Theme 1. Undergraduates and interns: Alvaro Garcia, TAMUK student worker. Dale Herrington, TAMUK student worker and summer intern with Ron Heiniger, NCSU Victor Gonzales, TAMUK student worker and summer intern at TAMUK Deanna Cano, TAMUK student worker and summer intern at TAMUK Amanda Lewis, TAMUK student worker and summer research assistant at TAMUK Kameron Koeppe, TAMUK student worker and summer intern with Ron Heiniger, NCSU Gregorio Carmona, summer intern with Kiersten Wise, Purdue How have the results been disseminated to communities of interest? Isakeit, T. 2014. Evaluation of atoxigenic strains of Aspergillus flavus for reduction of aflatoxin contamination of maize on commercial farms in Texas, USA. 36th Mycotoxin Workshop of the Society for Mycotoxin Research, Göttingen, Germany, June 18, 2014 Payne, G.A. 2014. Genomic Approaches to Characterize Regulator Circuits in Aspergillus flavus Controlling Aflatoxin Biosynthesis. Annual Meeting of the American Phytopathological Society, August 10-14, 2014. Minneapolis MN. Shim, W.-B.. Impact of Extreme Climate on Mycotoxin and Food Security. 3rd Korea-Japan Joint Plant Pathology Meeting, Pusan, South Korea. 2014. Woloshuk, C. P. Managing Molds and Mycotoxins in Grain. Post Harvest Workshop at the Purdue University Diagnostic Training and Research Center. West Lafayette, IN. August 21, 2014. Kim, M., Zhang, H., Woloshuk, C., Shim, W.-B. and B.-J. Yoon. Computational identification of functional network modules associated with the pathogenicity of Fusarium verticillioides. International workshop on computational network biology: Modeling, Analysis, and Control, Newport Beach, CA; 2014. Musungu, B., Obrain, R.G., Brown, R., Bhatnagar, D. Payne, G., Geisler, M., Fakhoury, A. M. 2014. Gene Regulatory Networks for Resistance in Maize to Ear Rots and to Aflatoxin Accumulation. Annual Meeting of the American Phytopathological Society, August 10-14, 2014. Minneapolis MN. Herrington, D., G. Schuster, A. Lewis, and P. Porter. Mycotoxin Levels Associated with Fall Armyworm (Spodoptera frugiperda) and Corn Earworm (Helicoverpa zea) Infested Corn. Undergraduate Poster competition. 2014 Subtropical Agriculture & Environments, Feb 21, 2014, Weslaco, TX Herrington, D., G. Schuster, A. Lewis, and P. Porter. Mycotoxin Levels Associated with Fall Armyworm (Spodoptera frugiperda) and Corn Earworm (Helicoverpa zea) Infested Corn. Undergraduate Poster competition (Won 3rd place). 2014 Annual Meeting of the Southern Branch of the American Society of Agronomy, Feb 2-4, 2014, Dallas, TX Dupnik, M., C. P. Woloshuk, G. L. Schuster, and S. D. Nelson. Assessment of Aspergillus flavus and Aflatoxin Production Under Low Oxygen Conditions. Undergraduate Poster competition. 2014 Subtropical Agriculture & Environments, Feb 21, 2014, Weslaco, TX Dupnik, M., C. P. Woloshuk, G. L. Schuster, and S. D. Nelson. Assessment of Aspergillus flavus and Aflatoxin Production Under Low Oxygen Conditions. Undergraduate Poster competition. 2014 Annual Meeting of the Southern Branch of the American Society of Agronomy, Feb 2-4, 2014, Dallas, TX What do you plan to do during the next reporting period to accomplish the goals? 1. Conduct a third year of field experiments with the goal of validating our previous results and finalizing region-specific recommendations for disease management with biocontrol applications. These results will provide the foundation for the new BMPs to reduce mycotoxin contamination and will be utilized to develop programs for disseminating these Extension-based research findings. Economic analysis of effective management practices will be conducted to provide recommendations for practices that consistently reduce mycotoxins in the most cost-effective manner. 2. Video resources will be developed that focus on ear rot identification, management, and fundamental information about mycotoxins. Videos will also be developed to showcase field protocols and results to show stakeholders how the research is being conducted. Videos will be concise, easily understood, and available on YouTube and our extension and research web sites (www.corntoxins.org and www.cornmycotoxins.com). We plan to film research and extension activities in Texas and Arkansas in year 3. Websites will be updated with new management practices and video content in year 3, and impact assessments of workshops and web resources will begin in year 3. 3. An Interactive and mobile device will be developed. This diagnostic tool will help producers and agribusiness personnel determine appropriate management recommendations for Aspergillus and Fusarium ear rots. The tool will be available in two formats: an internet-based interactive web portal and a smart phone App designed for Apple iOS mobile devices (iPhone and iPad). 4. We will complete our ddRADSeq genotyping of the year 1 isolates from across AR, IN, NC and TX. This will provide a preliminary estimate of the distribution of MAT1-1 and MAT1-2 mating types across the four states. 5. We will genotype progeny from field experiments to assess the capacity of fertilized sclerotia of A. flavus to subsequently form ascospores on soil. We will screen for putative females among strains that were isolated from corn and soil by setting up crosses whereby sclerotia of dominant genotypes are reciprocally fertilized to identify the female parent. 6. We will complete F. verticillioides isolations from year 2 ear samples and in a manner similar to A. flavus (Obj. 2.1), we will use ddRADSeq for population structure analysis. 7. We will determine if it is feasible to predict biomarkers in plants exposed to environmental stresses likely to increase risks for ear rot disease and higher levels of mycotoxin contamination. Critical for success is a good correlation between stress application and ear rot severity and mycotoxin levels at harvest. 8. We will continue to advance current transgenic lines toward the ultimate goal of obtaining homozygous lines that can be tested for reduced mycotoxin accumulation. 9. A leaf extracts assay will be used to evaluate transgenic plant lines for silencing. 10. New transgenic lines will be generated that target known aflatoxin or fumonisin biosynthetic and regulatory genes, as well as novel genes obtained from complementary efforts in the project. 11. New maize lines will be developed that contain constructs for specific silencing of GFP for visual assessment of HIGS in maize. These lines should help us address questions about tissue effects and cell-to-cell pathogen/host interactions. 12. With the developed algorithm, we will further analyze the data to identify subnetwork modules that are differentially expressed during times or conditions that correspond to mycotoxin biosynthesis. This approach will identify subnetwork modules that are (i) specifically or predominantly activated (or deactivated) during aflatoxin/fumonisin biosynthesis, (ii) differentially expressed during drought stress, and (iii) significantly different across corn hybrids B73 (moderately resistant) and 33K44 (susceptible). To improve specificity, we will filter out subnetwork modules that are associated with normal growth and development. 13. We will use the data generated in the different scenarios of the Best Management Practices (BMPs; Theme 1, informed by results from Themes 2 and 3) to determine the extent of aflatoxin and fumonisin reduction afforded in each scenario. 14. We will continue to attend US Environmental Protection Agency’s Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panels (SAPs), to learn more about how content area experts in RNAi technologies view the need to regulate these technologies in transgenic crops.

Impacts
What was accomplished under these goals? Our project objectives aim to equip corn producers with management practices and tools that will help reduce the risks of mycotoxin contamination. The project is divided into the four Themes. One of the activities of Theme 1 is to improve the best management practices (BMP) for corn farmers to reduce their ear rot and mycotoxin risks. Field experiments were conducted for a 2nd year in TX, IN, and NC to test and validate the BMPs. The work was presented to over 360 farmers during extension workshops. An extension website was established, which is easily assessable by mobile devices. We are updating the content to include disease identification, mycotoxin information, videos, and economic calculators that will help farmers answer questions about the BMPs. Research continued in this 2nd year to address the other Project Themes. To date, the impact of this research was new knowledge communicated through scientific presentations and publications. The aims of Theme 2 are to determine the impact of biocontrol application on the targeted fungal species (Aspergillus flavus) and to develop a new generation of biocontrol strains. This is a multiple year endeavor with thousands of fungal isolates being collected from experimental fields in NC, AR, IN and TX. This year, improvements were made in our analytical tool for genotyping the fungal populations, which increased robustness. Activity in Theme 3 research generated new transgenic corn lines expressing potential inhibiting RNAs that target at least 5 genes that impact mycotoxin production. We have also made progress towards finding new gene targets with our computer algorithms that model the functional association of fungal genes. A second year of greenhouse experiments were also conducted to correlate drought and heat stress with increased levels of aflatoxin and fumonisin contamination. One of the aims of Theme 4 is to determine the baseline for economic losses due to aflatoxin and fumonisin contamination in US corn. This was completed by averaging data on mycotoxin contamination levels from Iowa, Illinois, and Texas over three years; extrapolating to other corn-growing states in similar climatic regions; finding prices of corn for each state; and applying discount schedules for different uses of corn with differing mycotoxin levels. We estimated an average annual loss of $1.03-1.61 billion to the US corn industry.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Kim, M., Zhang, H., Woloshuk, C., Shim, W.-B. and B.-J. Yoon. 2014. Computational approach for identifying pathogenic modules of Fusarium verticillioides. IEEE/ACM Transactions on Computational Biology and Bioinformatics
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Miller, J. D., Schaafsma, A. W., Bhatnagar, D., Bondy, G., Carbone, I., Harris, L., Harrison, G., Munkvold, G., Oswald, I., Pestka, J., Sharpe, L., Sumarah, M., Tittlemier, S. and T. Zhou. 2014. Mycotoxins that affect the North American Agri-Food sector: state of the art and directions for the future. World Mycotoxin Journal 7 (1): 63-82.
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Olarte, R. A., Worthington, C.J., Horn, B.W., Moore, G.G., Singh, R., Monacell, J.W., Dorner, J.T., Stone, E.A., Xie, D-Y. and I. Carbone. 2014. Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus. PloS Pathogens
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Horn, B. W., Sorensen, R. B., Lamb, M. C., Sobolev, V. S., Olarte, R. A., Worthington C. J. and I. Carbone. 2014. Sexual reproduction in Aspergillus flavus sclerotia naturally produced in corn. Phytopathology 104:75-85.
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Olarte, R. A., Horn, B.W., Singh, R. and I. Carbone. 2014. Sexual recombination in Aspergillus tubingensis. Mycologia,
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Ridenour, J.B. and B.H. Bluhm. 2014. The HAP complex in Fusarium verticillioides is a key regulator of growth, morphogenesis, secondary metabolism, and pathogenesis. Fungal Genetics and Biology 69:52-64.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Wu, F. 2014. Global impacts of aflatoxin in maize: Trade and human health. World Mycotoxin Journal, (In press DOI:10.3920/WMJ2014.1737).
  • Type: Websites Status: Other Year Published: 2014 Citation: www.cornmycotoxins.com www.corntoxins.org
  • Type: Other Status: Published Year Published: 2014 Citation: Purdue Extension Bulletin BP-86-W. Diseases of Corn: Fusarium Ear Rot. C. Woloshuk and K. Wise. September 2014.
  • Type: Other Status: Published Year Published: 2014 Citation: Purdue Extension Bulletin BP-96-12-W. Diseases of Corn: Corn Disease Loss Estimates from the United States and Ontario, Canada-2012. Compiled by D. Mueller and K. Wise.
  • Type: Other Status: Published Year Published: 2014 Citation: Purdue Extension Bulletin BP-96-13-W. Diseases of Corn: Corn Disease Loss Estimates from the United States and Ontario, Canada-2013. Compiled by D. Mueller and K. Wise.


Progress 01/01/13 to 12/31/13

Outputs
Target Audience: Over the past year we have engaged growers in Indiana, Arkansas, Texas, and North Carolina. Much of the effort has been to explain the current knowledge about aflatoxin and biocontrol strategies, and to explain our effort to develop and improve management tools for reducing the risks of mycotoxin contamination. We also collected survey data from these clientele that accessed their knowledge and attitudes about mycotoxin risks, biocontrol strategies and GMOs. Research results were also presented to the scientific community at several professional meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Dale Herrington and Amanda Lewis, undergraduates from Texas A&M-Kingsville, received training from PIs at Texas A&M-College Station. These students will be involved in the fieldwork associated with Goal 1 experiments at Texas A&M-Kingsville. Megan A. Dupnik, a junior in Plant and Soil Science at Texas A&M Kingsville participated in summer (2013) research at Purdue University under the supervision of Drs. Wise and Woloshuk. Megan participated in research that determined the time required for aflatoxin M1 to clear the milk of goats that were fed aflatoxin-contaminated corn. Megan led the analysis of the milk, which involved purification of the aflatoxin M1 and its quantification by HPLC. Megan also participated in research to investigate the effects of low oxygen on Aspergillus flavus growth and aflatoxin production. This research was part of the on-going research on Purdue Improved Crop Storage (PICS) system. PICS consists of a low-cost bag system that creates a sealed barrier for store grain and results in a low-oxygen environment inside. Farmers in West and Central Africa are using PICS bags to control insect pests. Megan’s work was to determine if the PICS system could prevent the accumulation of aflatoxin during storage. Weiran Li is a PhD graduate student in the Department of Botany and Plant Pathology at Purdue University. Weiran’s research is focused on the project Goal 3.1 Establish biomarkers in corn that predict predisposition to mycotoxin contamination. Weiran’s academic advisor is Dr. Charles Woloshuk. Megan Meyers is a MSc student in Plant Pathology at North Carolina State University under the supervision of Drs. Carbone and Heiniger. Her research focuses on developing improved management strategies for reducing mycotoxins in corn (Goal 1). Megan Sexton is a PhD student co-majoring in Plant Pathology and Crop Science at North Carolina State University. Megan's research is focused on developing new biocontrol methods for mycotoxins (aflatoxin and fumonisin) in corn (Goal 2). Megan is co-advised by Drs. Carbone and Heiniger. Dr. Nicole Mitchell is receiving postdoctoral research training at Michigan State University (Dr. Wu). She is collecting and analyzing economic data and mycotoxin occurrence data for Theme 4 (Goal 4). How have the results been disseminated to communities of interest? A number of presentations were made during the year that included: Isakeit, T. “Management of aflatoxin with Afla-Guard and AF-36” 2013 Corn Disease Working Group Meeting, St. Louis, MO, Feb. 28, 2013. Isakeit, T. “Aflatoxin update” Upper Gulf Coast Feed Grain & Cotton Conference, Wharton, TX, Jan. 24, 2013. Isakeit, T. “Corn aflatoxin management update” Coastal Bend Crops Symposium, Corpus Christi, TX. Jan. 29, 2013. Isakeit, T. “Aflatoxin update” Mid-Season Crops Clinic, Thorndale, TX. April 19, 2013. Isakeit, T. “Aflatoxin management update” 2013 Fayette County Crops Tour, La Grange, TX, June 20, 2013. Isakeit, T. “Aflatoxin update” Fort Bend County Crops Tour, Rosenberg, TX, July 2, 2013. Schuster, G. “Mycotoxin Management: Now and Future Prospects”. Armstrong County Fall Producers Meeting, Claude, Texas Shim, W.-B. and Woloshuk, C.P. “Summer Research Opportunities on the Management of Aspergillus and Fusarium Ear Rots”. Texas A&M Kingsville. April 2013. This event was to recruit students into our summer research program. Wu, F. “A dietary shift to aflatoxin-free crops reduces liver cancer mortality.” Mycotoxins: Triple Threat to African Development, Washington, DC, February 2013. Wu, F. “Mycotoxin exposure and child growth impairment.” MAL-ED Project Meeting, Bill & Melinda Gates Foundation, Seattle, WA, May 2013. Wu, F. “Global impacts of aflatoxin: Trade and health.” International Aflatoxin-in-Maize Working Group: Global Solutions for Worldwide Problems, New Orleans, LA, May 2013. Wu, F. “A tangled web: Aflatoxin regulations and social networks of food trade worldwide.” MycoRed Europe, Martina Franca, Italy, May 2013. Wu, F. “The global burden of disease caused by food contaminants.” Summer Academy in Global Food Law & Policy, Granada, Spain, July 2013. Wu, F. "Improving Food safety for Human and Animal Health: Lessons from Infectious Disease Control." GRF One Health Summit. Davos, Switzerland, November 2013. What do you plan to do during the next reporting period to accomplish the goals? A second year of experiments will be conducted in IN, NC and TX to address questions about the effectiveness of timing and rate of application of Afla- Guard and AF36, effectiveness of Bt hybrids and transgenic hybrids with drought-tolerant traits on aflatoxin and fumonisin contamination reduction, and the interactions of fungicides and biocontrol. Field demonstrations and winter workshops will be organized in each state to improve clientele awareness of corn ear rots and mycotoxin issues. Begin development of an interactive and mobile device designed to be a diagnostic tool to help producers and agribusiness personnel determine appropriate management recommendations for Aspergillus and Fusarium ear rots. Continue the analysis of effect of biocontrol strain application on the population structure of A. flavus in AR, IN, NC, and TX, which we initiated in 2013, and complete the second year of strain isolations from soil samples. A. flavus isolates from soil and corn ears, including ascospore progeny isolates, will be analyzed for aflatoxins and grouped based on aflatoxin/CPA chemotypes. Continue the population structure analysis for F. verticillioides and complete strain isolations from ear samples during year 2. Tissues collected biomarker discovery will be analyzed by qPCR to examine the expression of a set of known genes associated with disease resistance and stress responses. We also will analyze for soluble sugar and free amino acids. The greenhouse experiment will be repeated in the summer of year 2 with a Midwest hybrid. F. verticillioides and A. flavus transcripts from multiple kernel samples (from in-field and in-lab inoculation) will be sequenced at Texas A&M. Specifically, at North Carolina State, we performed field inoculation with A. flavus and F. verticillioides using a pinbar on B73 and hybrid N78S-3111 (R3~R4). Make estimates of how much corn growers in selected states are currently losing economically due to excessively high mycotoxin levels in their corn. For both the RNAi transgenic corn lines developed in Theme 3 and next-generation biocontrol developed in Theme 2, we will compile and describe the potential environmental impacts, and how these and potential human health impacts would be subject to regulatory review and oversight in the United States.

Impacts
What was accomplished under these goals? 1. Design and deliver best management practices for mycotoxin reduction. Experiments were conducted in Indiana Texas and North Carolina that addressed three areas: 1) the effectiveness of application timing and rate of Afla-Guard and AF-36 on reducing mycotoxin contamination, 2) the impact of Bt hybrids on aflatoxin and fumonisin levels in corn, 3) the interactions between foliar-applied fungicides and biocontrol agents. Information on ear rots and mycotoxins was presented at 16 Extension programs in Indiana, Texas, and North Carolina. These programs focused on ear rot identification, in-season management strategies, understanding mycotoxin development, and mycotoxin management in stored grain. Attendees included certified crop advisors, industry representatives, custom applicators, and farmers. 2. Develop next generation of biological control. A pilot study was completed that determined the most effective methodology for adequately sampling the genetic diversity of A. flavus on harvested corn ears. Samples of 10 corn ears each were collected from 2012 fields located in North Carolina (NC), Indiana (IN) and Arkansas (AR). After incubation for 3 days at 37 C, A. flavus isolates were collected and genotyped for mating type and variation at a microsatellite locus (AF17) to determine if kernel-plating method influences the measure of genetic diversity. Variation at AF17 was used for clone correcting MAT genotypes; AF17 is highly variable and useful for haplotype correction. Results from mating experiments showed that MAT1-1/MAT1-2 individuals are functionally heterothallic in a dose-dependent manner. Based on these results it was determined that it is best to increase the sample size to make sure we recover the underlying population structure. Field experiments in AR, IN, TX, and NC to determine the impact of biocontrol agents on native population structure were started. Prior to biocontrol applications in each location, soil samples were collected. A total of 3840 strains are being sent to NC for genotyping. NC developed a two-enzyme genotyping-by-sequencing approach to produce high-quality polymorphism data for 1) monitoring changes in the genetic/genomic architecture of applied biocontrol strains, 2) providing accurate estimates of haplotype diversity, and 3) performing detailed population genetic studies. 3. Develop biomarkers for host susceptibility & generate RNAi-based transgenics for resistance. Towards the identification of biomarkers, greenhouse experiments were performed.. Growth characteristics of the selected corn hybrid were established. Plant height, leaf number, and time to silking and ear maturation were determined. Ear inoculation tests comparing the various techniques were done to establish disease by both A. flavus and F. verticillioides. Stress (temperature and drought) was applied on the eighth week at planting. Measurements were taken weekly on leaf water potential, photosynthesis, leaf surface temperature, and soil moisture. Tissue was collected for amino acid, soluble sugar, and gene-expression analyses. Host Induced Gene Silencing (HIGS) constructs were generated for initial testing in A. flavus and F. verticillioides. A total of 17 gene constructs were made. Fungi containing these constructs are being tested for reduced mycotoxin production. Plant silencing constructs were made for six of the genes. Amylase gene RNAi corn lines were generated by Agrobacterium-mediated transformation of immature embryos. Plants and seed for these lines were obtained. Transgenic calli for other five silencing constructs are being regenerated. Plantlets are being picked and propagated. An RNA-Seq dataset from three strains of F. verticillioides (wild type, and two fumonisin-deficient mutants) grown on a synthetic medium was used to establish protocols for network modeling and regulatory logic prediction. Time-course experiments were conducted to obtain infected tissues of A. flavus and F. verticillioides during the infection of kernels. 4. Assess risks, regulatory issues, and socioeconomic impacts . Baseline data were collected on levels of aflatoxin and fumonisin in maize for selected regions and states of the US. Of interest were regions where Bt corn hybrids were widely planted, which have been shown to have significantly lower fumonisin levels compared to non-Bt isolines; less consistently, Bt hybrids have been shown to lower aflatoxin levels as well.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Horn, B. W., Sorensen, R. B., Lamb, M. C., Sobolev, V. S., Olarte, R. A., Worthington C. J. and I. Carbone. 2013. Sexual reproduction in Aspergillus flavus sclerotia naturally produced in corn. Phytopathology (In Press).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Isakeit, T. Evaluation of atoxigenic strains of Aspergillus flavus for aflatoxin control in corn on commercial farms in Texas. Phytopathology 103(Suppl. 2):S2.65.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Li, W. and Woloshuk, C. P. Aspergillus flavus in corn ears from Indiana under the drought conditions in 2012. Phytopathology 103(Suppl. 2):S2.80.