Source: PENNSYLVANIA STATE UNIVERSITY submitted to
MYCOTOXINS: BIOSECURITY, FOOD SAFETY AND BIOFUELS BYPRODUCTS (NC129, NC1025)
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0223928
Grant No.
(N/A)
Project No.
PEN04418
Proposal No.
(N/A)
Multistate No.
NC-_OLD1183
Program Code
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Project Director
Kuldau, GR.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Plant Pathology & Environmental Microbiology
Non Technical Summary
The most important relevant issues facing grain and livestock producers are preventing mycotoxin contamination of food and feed, and reducing the deleterious effects of mycotoxins on livestock. For grain buyers and food processors, the primary need is a reliable method for rapid assessment of grain quality pertaining to mycotoxins and mycotoxigenic fungi. Rapid methods to detect mycotoxins at the first points of sale (elevators) as well as methods to detect mycotoxigenic fungi in the commodity (e.g. DON-producing Fusarium in barley) would help to address these concerns. Finally, workers who are responsible for animal and human health need information about the toxicity, carcinogenicity, modes of action, and biomarkers of exposure and disease for all categories of mycotoxins. The proposed research is wide-ranging and could lead to negative consequences if not completed. First, the presence of mycotoxins is an important health hazard. Hazard assessments including exposure assessment and evaluation of toxicity are essential. We propose basic research to define the toxicity of several important mycotoxins. Without this information, it is impossible to assess risks associated with mycotoxins. Additionally, the presence of mycotoxins in grain is an economic concern, especially in the context of global markets. Without an aggressive research program to prevent, treat, and contain outbreaks of mycotoxins in grain, US grain producers will suffer the consequences of reduced marketability of their products. Furthermore, the proposed research addresses biosecurity concerns. The natural occurrence of mycotoxins in grain is an important security concern for the grain industry and end-users of grain; mycotoxins have been used as agents of terrorism, e.g. aflatoxin in Iraq. Without a proactive research program to find innovative ways to monitor, prevent, and treat mycotoxin contamination, US agriculture will be unprepared to deal effectively with a mycotoxin outbreak, regardless of its origin. Finally, the production of mycotoxins by mycotoxigenic fungi represents a continuing problem in agriculture. Improving our understanding of how mycotoxin biosynthesis is regulated in grain-associated fungi will not only lead to novel treatment strategies, but will also advance our understanding of fungal pathogenesis in general. The scientists involved in this multistate, multidisciplinary research proposal work individually on mycotoxin issues related to their respective disciplines and areas of expertise. Just as agriculture is diverse and varies greatly from state to state, the occurrence and severity of mycotoxin outbreaks vary widely across the US. A multistate effort ensures a thorough approach to investigate a complex and highly variable phenomenon that has significant impacts on both producers and consumers. Outputs will include information on the action of mycotoxins in livestock and animal models. This information will be applicable to the risk assessment process. The work will also address stakeholders' continuing need for new detection and monitoring methods for grain and in DGs.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121510110215%
2121542110215%
3141510110220%
3141542110220%
7121510110215%
7121542110215%
Goals / Objectives
1. Develop data for use in risk assessment of mycotoxins in human and animal health. 2. Establish integrated strategies to manage and to reduce mycotoxin contamination in cereal grains and distillers grains. 3. Define the regulation of mycotoxin biosynthesis and the molecular relationships among mycotoxigenic fungi. Outputs: * Refereed journal publications; many will be co-authored by the members from multiple states. * Development and validation of new management tools for diagnosis and prevention of mycotoxin contamination. * Transfer of valuable research information to clientele groups (industry, government, grain producers and food producers) through general publications, website, and extension programs.
Project Methods
2.1 Surveillance Surveillance for mycotoxins and mycotoxigenic fungi is critical for security as well as for maintaining high quality grains and grain products. PCR-based methods to detect mycotoxigenic fungi have provided rapid and inexpensive alternatives to techniques based on morphology. Committee members from TX, IN, PA, ND will collaborate to expand the use of this PCR identification technique and related DNA technologies for surveillance. Others will continue to develop more multiplex assays from mycotoxin-specific genes. We will also work with university diagnostic labs to implement these techniques. 2 3.4 Understanding the cause for high mycotoxin in asymptomatic grains PA will quantify mycotoxin levels in grains at different developmental stages and under different conditions of growth of the host plants. Milestones: (2010): We will expand the content at our website and improve the links. (2012): We will organize a mycotoxin symposium at the Midwest AOAC annual meeting. (2014): We will organize a mycotoxin symposium at the Midwest AOAC annual meeting.

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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Deoxynivalenol is the mycotoxin to which humans in North America are most frequently exposed primarily through consumption of wheat-based products. Contamination occurs when Fusarium Head Blight disease caused by the fungus Fusarium graminearum occurs on wheat. We have been conducting experiments to shed light on the phenomenon where grain that appears visually sound and has acceptable test weight can contain levels of deoxynivalenol (DON) above 2 ppm. At this level millers and bakers must find and purchase supplementary wheat with no detectable DON to achieve acceptable product. Wheat plants are susceptible at flowering and up to two weeks after flowering. One hypothesis is that cooler than average temperatures during the flowering time can result in reduced growth of the pathogen, F. graminearum, but increased rates of toxin production. In a previous experiment our observations were that this could happen. We are repeating this work to corroborate or refute this initial observation. Specifically, we have conducted a growth chamber experiment with flowering wheat plants that were inoculated with F. graminearum. Inoculated plants were grown at two temperatures 15 C and 22 C to compare the levels of DON produced relative to fungal biomass at each temperature. Plants were started in the greenhouse and transferred to growth chambers at flowering. Inoculations were made to a single central spikelet on the wheat head at 50% anthesis, and florets were harvested two-day intervals up to grain maturity. Extraction of florets for DON and ergosterol, a marker of fungal growth, have been done for approximately half of the sample set, and these extracts have been analyzed by gas chromatography with electron capture detection. In recent years there has been increased concern amongst growers, buyers, millers and other agriculture professionals about apparent increased DON levels in wheat and corn produced in Pennsylvania. We conducted a survey of F. graminearum isolated from corn and wheat debris in the state. We confirmed species by sequencing a portion of the elongation factor one alpha gene and comparison of the sequences with the Fusarium ID database. We then determined the DON chemotype of each isolate. Reports from other areas of the United States and Canada have demonstrated that the 3-acetyl-deoxynivaleol (3-ADON) chemotypes tend to produce overall higher levels of DON than the 15-acetyl-deoxynivalenol types. Of the 95 F. graminearum isolates examined all but three were the 15-ADON type suggesting that an incursion of the 3-ADON type in Pennsylvania is not a likely explanation for the apparent increase in DON levels in wheat and corn produced in the state. We have initiated a new project to isolate microbes from soil, wheat heads and corn silage that possess the ability to transform deoxynivalenol or are inhibitory to F. graminearum. Our approach is to inoculate the above samples into a mineral salts medium containing deoxynivalenol as a sole carbon source. Initial cultures are diluted in fresh medium over three rounds. This will require a large amount of deoxynivalenol for use in the mineral salts medium. The cost of purchasing this material from commercial suppliers is prohibitive. As such we have collaborated with Dr. George Rottinghaus at the University of Missouri Veterinary School an expert on analysis and isolation of mycotoxins. A known high DON producing strain of F. graminearum was grown on autoclaved rice media. PhD student Anita Behari extracted and analyzed this material in Dr. Rottinghaus' lab with his assistance and oversight. We now have in hand a supply of deoxynivalenol to begin this work. The maize pathogen and endophyte Fusarium verticillioides produces the mycotoxin fumonisin that occurs in ears, stalks and leaves of the plant. Fumonisins are known to cause fatal diseases in livestock and are correlated with esophageal cancer and neural tube defects in humans. Through analysis of the genome sequence of F. verticillioides we identified two genes responsible for the production of or regulation of reactive oxygen species. FvNoxA has homology to other fungal NADPH oxidases, and FVNoxR has homology to other fungal regulators of NADPH oxidases. Strains with disruptions in these genes were non-pathogenic on maize ears, stalks and seedlings and showed several developmental growth defects. Production of fumonisin was significantly lower in these strains than in the non-disrupted wild type strain.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, G., Blatt, A. Z., Geiser, D. M., Jimenez-Gasco, MM, and Kuldau, G. A. 2015. Mating type and spore killing characterization of Fusarium verticillioides strains. Mycological Progress 14:16.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, G. Wenner, N., and Kuldau, G. A. 2015. FvSO regulates vegetative hyphal fusion, asexual growth, fumonisin B1 production and virulence in Fusarium verticillioides. 2015. Fungal Biology, doi:10.1016/j.funbio.2015.08.013.


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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We will be analyzing two sets of samples for deoxynivalenol and deoxynivalebol-3-glucoside levels and for one sample set ergosterol levels as well as a measure of fungal growth. The first set of samples are from a previous field experiment. We will be looking at levels of deoxynivalenol and deoxynivalenol-3-glucoside at multiple time points from anthesis to grain maturity to learn about development of the masked mycotoxin deoxynivalenol-3-glucoside in different wheat varieties and over time. Currently we are working to re-establish a gas chromatography method for this purpose. The second of samples are from a growth chamber experiment that addresses the question of the impact of ambient temperature on in planta deoxynivalenol production relative to fungal growth measured by ergosterol. We have previous results indicating that lower temperatures are associated with higher levels of deoxynivalenol production relative to fungal biomass. This experiment will confirm or deny those previous observations.

Impacts
What was accomplished under these goals? We have conducted a growth chamber experiment with flowering wheat plants that were inoculated with F. graminearum the causal agent of wheat Head Blight Disease and a producer of the mycotoxin deoxynivalenol. Inoculated plants were grown at two temperatures, 15 C and 22 C to compare the levels of deoxynivalenol produced relative to fungal biomass at each temperature. Plants were started from seed in the greenhouse, grown to flowering then transferred to growth chambers for controlled temperature conditions. Inoculations were carried out at 50% anthesis, and florets were harvested at two day intervals up until grain maturity.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Baines, D., Sumarah, M., Kuldau, G., Juba, J., Mazza, A., Masson, L. 2013. Aflatoxin, fumonisin and shiga toxin-producing Escherichia coli infections in calves and the effectiveness of Celemanax r/Dairymans Choice T applications to eliminate morbidity and mortality losses. Toxins 5(10): 1872-1895.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Rahman, A., Kuldau, G. A., Uddin, W. 2014. Induction of salicyclic acid-mediated defense response in perennial ryegrass against infection by Magnaporthe oryzae. Phytopathology104:614-623.


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We plan to analyze a set of grain samples including six wheat cultivars for the presence of deoxynivalenol and the “masked mycotoxin” deoxynivalenol by chromatography. These samples were harvested at the time of inoculation, 5, 10, 15, 20 days post-inoculation and at maturity. We expect data collected will provide insight into the dynamics of deoxynivalenol formation in the field.

Impacts
What was accomplished under these goals? There is increased concern amongst growers, buyers, millers and other agriculture professionals about apparent increased levels of the mycotoxin deoxynivalenol in wheat and corn produced in Pennsylvania. We conducted a survey of the wheat head scab organism, F. graminearum that produces deoxynivaneol, to determine the incidence and prevalence of the more toxigenic 3-acetyl-deoxynivalenol strains in Pennsylvania. Corn and wheat debris were collected from fields at 75 locations in 18 PA counties in January through March 2013. Five pieces per site were plated on medium selective for the fungal genus Fusarium. Individual pure cultures created from these plates resulted in nearly 400 cultures. All but a few were determined visually to be Fusarium. The species of the cultures was determined by amplification of he five prime end of the EF1-alpha gene followed by DNA sequencing and comparison the sequence to the Fusarium ID database. About ¼ of the cultures (95) were identified as F. graminearum the causal agent of head scab of wheat and ear and stalk rot of corn. Of these 95 all were of the 15-acetyl-deoxynivalenol chemotype except 3 isolates that were confirmed to be the 3-acetyl-deoxynivalenol type. This data indicates that an incursion of the more toxigenic 3-acetyl-deoxynivalenol strains into PA is not a likely explanation for the apparent increase in overall deoxynivalenol levels in wheat and corn.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Malcom, G. M., Kuldau, G. A., Gugino, B. K., Jimenez-Gasco, M. D. Hidden host plant associations of soilborne fungal pathogens: An ecological perspective. Phytopathology, 103: 538-544.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Geiser, D. M., T. Aoki, C. W. Bacon, S. E. Baker, M. K. Bhattacharyya, M. E. Brandt, D. W. Brown, L. W. Burgess, S. Chulze, J. J. Coleman, J. C. Correll, S. F. Covert, P. W. Crous, C. A. Cuomo, G. S. De Hoog, A. Di Pietro, W. H. Elmer, L. Epstein, R. J. Frandsen, S. Freeman, T. Gagkaeva, A. E. Glenn, T. R. Gordon, N. F. Gregory, K. E. Hammond-Kosack, L. E. Hanson, M. Jimenez-Gasco Mdel, S. Kang, H. C. Kistler, G. A. Kuldau, J. F. Leslie, A. Logrieco, G. Lu, E. Lysoe, L. J. Ma, S. P. McCormick, Q. Migheli, A. Moretti, F. Munaut, K. ODonnell, L. Pfenning, R. C. Ploetz, R. H. Proctor, S. A. Rehner, V. A. Robert, A. P. Rooney, B. Bin Salleh, M. M. Scandiani, J. Scauflaire, D. P. Short, E. Steenkamp, H. Suga, B. A. Summerell, D. A. Sutton, U. Thrane, F. Trail, A. Van Diepeningen, H. D. Vanetten, A. Viljoen, C. Waalwijk, T. J. Ward, M. J. Wingfield, J. R. Xu, X. B. Yang, T. Yli-Mattila, and N. Zhang. 2013. One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use. Phytopathology 103 (5):400-8.


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: We are studying the growth of microorganisms on air conditioning cooling coils. Samples from two coils, one in a recreation building at Penn State and one in a classroom and office building at the University of Tampa, by removing the material on the surface and in between the fins of the coil. This material is then mixed with water and plated on five types of media to select for bacteria and different types of fungi. Individual colonies are then picked and purified to single organisms. These cultures are then subjected to direct PCR of the 16S rDNA for bacteria and 18S rDNA for fungi and the resulting amplicons are then sequenced. Sequences obtained are compared to sequence databases and the best match is the basis for identification. A wide variety of bacteria and fungi have been found thus far. In addition to the fungal genera commonly found in indoor air such as Cladosporium and Chaetomium we have found a variety of yeast species and basidiomycete genera. A variety of Gram positive and Gram negative bacteria have been found including some tentatively identified as human pathogens. PARTICIPANTS: Dr. Gretchen Kuldau is the principal investigator. She has been assisted by Ms. Nancy Wenner, a research associate and Mr. Edward Kaiser. She has provided supervisory scientific guidance to Mr. Li Guo (Ph. D. student during part of the reporting period). These individuals have executed the experiments analyzed the data and prepared reports on the findings. TARGET AUDIENCES: Target audiences are architectural engineers, industrial hygienists and other scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The data collected increases the knowledge regarding microorganisms in the indoor environment. Specifically, this provides information regarding sources of indoor microbes.

Publications

  • No publications reported this period


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: We are studying the maize pathogen and endophyte Fusarium verticillioides and are interested in identifying genes involved virulence. Homologs of three genes identified in other fungi were identified in the genome sequence of F. verticillioides. These genes are involved in hyphal fusion and generation and regulation of reactive oxygen species production. Strains of F. verticillioides with disruptions in each of the genes were made previously using DNA transformation procedures. Strains with a disruption of the gene involved in hyphal fusion were non-pathogenic on maize ears, stalks and seedlings and showed several development growth defects. Overall conidial production was decreased and the average conidial size was decreased compared to wild type. Additionally, these isolates grew more slowly than wild type. Strains with disruptions in either a gene encoding a NADPH oxidase or a gene regulating NADPH oxidases had significantly reduced pathogenicity on maize ears, stalks and seedling but showed normal in vitro growth rates. Production of the mycotoxin fumonisin was significantly lower than wild type in all three of the disruption strains. PARTICIPANTS: Dr. Gretchen Kuldau is the principal investigator. She has been assisted by Ms. Nancy Wenner, a research associate. She has provided supervisory scientific guidance to Mr. Li Guo (Ph. D. student during the reporting period). These individuals have executed the experiments analyzed the data and prepared posters for presentations at national meetings. TARGET AUDIENCES: Target audiences are agriculture professionals in the grain and feed industry, extension educators, government regulatory agencies, extension specialists, breeders and mycotoxin and other scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The data collected during these experiments increases the overall knowledge of virulence mechanisms of F. verticillioides. While fumonisin mycotoxins have been previously shown to have a role in virulence this is only for seedling blight pathogenicity. These results show that some genes, and their products are involved with all of the three types of disease this fungus causes on maize namely seedling blight, ear rot and stalk rot. The new knowledge generated indicating a role for reactive oxygen species in F. verticillioides pathogenicity on maize opens new opportunities for deeper understanding of virulence mechanisms and potential targets for control.

Publications

  • Hallen-Adams, H., N. Wenner, G. Kuldau, and F. Trail. 2011. Deoxynivalenol gene expression during wheat kernel colonization by Fusarium graminearum. Phytopathology 101:1091-1096.
  • Baines, D., S. Erb, R. Lowe, K. Turkington, E. Sabau, G. Kuldau, J. Juba, and R. A. Roberts. 2011. Mouldy feed, mycotoxins and Shiga-toxin-producing Escherichia coli colonization in associated with Jejunal Hemorrhage Syndrome in beef cattle. BMC Veterinary Research 2011. 7:24.