Source: KANSAS STATE UNIV submitted to
MYCOTOXINS: BIOSECURITY, FOOD SAFETY AND BIOFUELS BYPRODUCTS (NC129, NC1025)
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1007402
Grant No.
(N/A)
Project No.
KS00-0006-NC1183
Proposal No.
(N/A)
Multistate No.
NC-_old1183
Program Code
(N/A)
Project Start Date
Oct 1, 2015
Project End Date
Sep 30, 2020
Grant Year
(N/A)
Project Director
Leslie, J.
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Plant Pathology
Non Technical Summary
Mycotoxins and the fungi that produce them are major health hazards and commercial uses of contaminated materials are limited. Identification of the fungi that produce these toxins remains difficult, and there are significant taxonomic discrepancies that remain to be resolved. Improving identification requires research to identify significant species and subgroups within species and educational efforts to train scientists and plant diagnosticians how to identify these significant groups. In addition, we will maintain critical strains for long-term access to them from the fungal research community. The availability of these strains allows research results to be readily standardized as effects of genetic background can be minimized. Most strains of Fusarium have the genetic capacity to synthesize multiple secondary metabolites, many of which are mycotoxins. By analyzing progeny of an inter-species cross we can identify strains that produce novel combinations of these secondary metabolites and determine if the co-production of the metabolites changes the pathogenicity or toxicity of the strains towards their plant hosts.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2062299106033%
7121520106034%
7121540106033%
Knowledge Area
206 - Basic Plant Biology; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
2299 - Miscellaneous and new crops, general/other; 1540 - Hard red winter wheat; 1520 - Grain sorghum;

Field Of Science
1060 - Biology (whole systems);
Goals / Objectives
Objective 1: Develop data for use in risk assessment of mycotoxins in human and animal health. Objective 2: Establish integrated strategies to manage and reduce mycotoxin contamination in cereals and in forages. Objective 3. Better Understand the Biology and Ecology of Mycotoxigenic Fungi.
Project Methods
Accessioned strains in the Fungal Genetics Stock Center will be preserved in at least two different manners: on anhydrous silica gel at 4C, freeze dried and stored in ampules at 4C, as spore/hyphal fragment suspensions in 15-25% glycerol suspensions at -80C, or in liquid nitrogen.Non-accessioned strains will be preserved on anhydrous silica gel at 4C or as received from the depositor.Fusarium Laboratory Workshops have been held under Dr. Leslie's supervision since 2000. His group has developed a standard format that flexes to allow the introduction of new ideas and topics while retaining a central core of information. Since 2002, the workshops have been held in odd years at various international locations (Argentina, Australia, Italy, Malaysia, South Africa and South Korea) to ensure that scientists who might never come to the United States have the opportunity to participate in these workshops. Workshops outside the United States follow the same general format as those conducted within the US. Workshops at Kansas State University are offered in June of even numbered years. Material covered includes morphological and molecular approaches to taxonomy and identification, mycotoxin biosynthesis and detection, genetic and genomic issues, population genetics and variation, and epidemiology, amongst others. The general format and timing of these workshops is expected to continue.Genetic variation is being assessed with various molecular tools.Most commonly, Amplified Fragment Length Polymorphisms (AFLPs) have been used.This protocol uses DNA that has been digested to completion with two different restriction sites.Relatively short (~20 bp) linkers of known sequence are annealed to the restricted DNA, which is then amplified via PCR with primers that are based on the linker sequences.Primers for the linker associated with one of the restriction enzyme sites are labeled with 32P.Amplified fragments are separated on acrylamide sequencing gels and the size of labeled fragments determined following autoradiography.Each band is assumed to represent a single locus, with bands between 200 and 500 bp scored preferentially.The resulting multi-locus data are used for population genetic analyses facilitated by widely available software for determining population structure, genetic polymorphism, migration and related standard parameters.The data also are used to assess sexual fertility and to determine the relative frequency of sexual and asexual reproduction in the population.Inter-hybrid crosses are made on carrot agar following standard protocols for Fusarium spp.Fertility is low and no more than 10-20 spores are collected from any single perithecium.Genetic segregation amongst current progeny is analyzed via GBS.Recombination maps are generated for phenotypes of interest relative to molecular markers.Regions of interest are identified on the physical map by using the molecular polymorphisms to correlate the recombination and physical maps.Open reading frames within these regions are evaluated to determine the probability that they could be responsible for the observed segregating phenotype.The parental strains for the cross have been sequenced and allow straightforward localization of the genetic results when the number of progeny evaluated is large enough for the map resolution required.Analysis of feeds for mycotoxins will be performed by liquid chromatography/mass spectroscopy/mass spectroscopy.

Progress 10/01/15 to 09/30/20

Outputs
Target Audience:Scientists working with fungi, mycotoxins and the substrates they contaminate. Includes US and host country professional colleagues and staff for the USAID sponsored Feed the Future Lab for the Reduction of Post-Harvest Losses, and for the European Union Horizon 2020 Projects MyToolBox and MycoKey Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Fusarium Laboratory Workshops have been offered annually since 2000, with over 700 participants from 71 different countries. The Workshop scheduled for summer 2020 at Northwest Agricultural University in China has been postponed indefinitely as has the 2021 Workshop scheduled to occur in Manhattan, Kansas. Before each workshop in Manhattan, we allow 2-4 students and 1-2 individuals who will be organizing the next succeeding year's conference to participate in the week before preparations to learn what is involved in organizing and managing a conference such as this one. How have the results been disseminated to communities of interest?Fusarium Laboratory Workshop presentations Presentations at professional meetings and publications in professional journals. Nominal group discussions have been critical parts of Feed the Future final project meetings and were embedded in both the MyToolBox and MycoKey Horizon 2020 projects. In the Feed the Future and MyToolBox sessions, there were multiple scientific presentations aimed at an educated general audience followed by the structured discussions. MycoKey convened "roundtable" format discussions with only scientific experts participating in the discussions. Results of several discussion sessions have been published to provide advice to policy makers in Europe and China. In both Nepal and Afghanistan the discussion results were important for generating local interest in mycotoxins as a health and economic issue and as way to involve USAID mission personnel in solving the problem and connecting to local leaders on the issue. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? What was accomplished Impact -The Fungal Genetics Stock Center remains a major resource for the science community. It has provided continuity for the availability of critical strains and other genomic resources since 1960. The FGSC survives on very little funding (fees only) which covers the cost of a technician, postage and operational materials. In the last five years the FGSC has distributed approximately 3000 individual strains and approximately 1500 96-well plates of strains. These materials drive research across all fungi in the United States and over 50 other different countries. Research conducted has led to ~ $2.2 million in funding from USAID on mycotoxins in Nepal, Honduras and Afghanistan as threats to health, especially of children, and to food and economic security. These efforts have laid the groundwork for future work in these areas, with extensive basic survey work conducted to determine levels of aflatoxins, fumonisins, ochratoxin A and ergot present in foods consumed by the local population. Strategies appropriate for local activitiesto reduce toxin contamination were identified and guidance for implementation provided. 2019-2020 activities Fungal Genetics Stock Center (FGSC) -The FGSC's activities have been reduced to the minimal tasks of supplying strains, plasmids, media and genomic DNA to scientists who request them. Additional strains are accessioned as people provide them. A major effort has been to digitize the strain deposit sheets and make the available through the K-State library's archival storage system - K-Rex. At this time all of the deposit sheets for strains have been scanned and a small subset have been formatted for archival recovery information. The paper documents occupy between 40 and 50 linear feet of shelf space and are not particularly easy to access. Kevin McCluskey, the curator of the FGSC when it moved to K-State in 2014, has left the university for a position in private industry. It is unlikely that he will be replaced in the coming year. Fusaric and fusarinolic acid production -Fusaric acid is an unregulated mycotoxin produced by most species of Fusarium. In F. temperatum and F. subglutinans it appears to be an intermediate in the biosynthesis of fusarinolic acid, a compound of uncertain toxigenicity. The amount of fusarinolic acid produced always exceeded the amount of fusaric acid produced (max 50,000 ng/g and 4,500 ng/g, respectively). Temperature, aW, incubation time, and the two- and three-way interactions amongst them all significantly impacted fusaric acid and fusarinolic acid accumulation. The highest level of FA was detected at 0.995aW and was independent of temperature and length of incubation, suggesting that there is a limit to the amount of fusaric acid that can be accumulated, at least under laboratory growth conditions. Strikingly high amounts of fusarinolic acid were observed under all incubation conditions, often exceeding fusaric acid levels by 20- to 200-fold. This result suggests that fusarinolic acid is more important to the fungus than is fusaric acid. The role of the accumulated fusarinolic acid is unknown, but its role as a toxin may have been discounted since studies to date report limited toxicity. However, if fusarinolic acid is tested for toxicity at higher levels, then it could have significant toxicological, or other effects that have not previously been considered. Fumonisin and beauvericin production by F. subglutinans and F. temperatum -F. subglutinans and F. temperatum are common maize pathogens whose ability to produce beauvericin and fumonisin mycotoxins is not settled, as reports of toxin production are not concordant. We determined both the chemotypes and genotypes for 25 strains from both species.None of the strains examined from either species produced fumonisins. These strains lacked fum biosynthetic genes, but retain homologs of some genes that flank the fum gene cluster in Fusarium verticillioides. None of the 13 F. subglutinans strains we examined produced beauvericin although 9 of 12 F. temperatum strains did. A complete beauvericin (Bea) gene cluster was present in all the three complete genome sequences examined. The Bea1 gene was presumably functional in F. temperatum but was not functional in F. subglutinans due to a large insertion and multiple mutations that resulted in premature stop codons. The accumulation of only a few mutations expected to disrupt Bea1 suggests that the process of its inactivation is relatively recent. Variation in the ability of strains of F. temperatum to produce beauvericin requires further study. Toxin production on maize, sorghum and pearl millet cuturedin vitro- We recovered 18 Fusarium strains from maize (n=10), sorghum (n=7) and pearl millet (n=1) growing in the same field in Nigeria. The 17 strains from maize and sorghum were all F. proliferatum and the one strain from pearl millet was F. pseudonygamai. Under conducive laboratory conditions, the 17 F. proliferatum strains produced fumonisins, 11 in relatively large quantities (700-17,000 mg total fumonisins per kg of culture material), and six at <45 mg/kg. Ten F. proliferatum strains also produced >100 mg of moniliformin per kg culture material with a maximum of 8,900 mg/kg culture material. All strains could use all grains for growth and toxin production, regardless of the host from which they were isolated. Isolates varied in the amount of toxin produced on each substrate, with toxin production a property of the strain and not the host from which the strain was recovered. However, the extent to which a toxin-producing phenotype could be altered by the grain on which the fungus was grown is consistent with subtle genetic × environment interactions that require a larger data set than the one we analyzed rigorously identify. If the amount of toxin produced on the various grains in this study reflects real-world settings, e.g., poor storage, then the consumers of these contaminated grains could be exposed to mycotoxin levels that greatly exceed the tolerable daily intakes. Communications strategy -Talking about mycotoxins amongst scientists often is easy, as the potential consequences are viewed from a perspective that breeds familiarity with potentially serious health problems. However, translating objective results into information that can be used profitably by non-scientists often is a difficult task. Experts in communications also have a difficult time with the mycotoxin issue because they lack the technical background to understand the causes, consequences and potential remedies associated with mycotoxin contamination. In collaboration with K-State's Vice-President for Communications and Marketing, we developed a framework that can be used by both scientists and communicators as they think about how to convey potentially scary food safety information to the public without causing a panic in the process. The framework was developed with less-developed countries in mind, but can also be used in developed countries. The major concerns people have about mycotoxin contamination are health- and financial-related. Stakeholders along the food chain have different backgrounds and interests and must be identified and grouped for effective communications, i.e., "one size fits all" is not a good strategy. Similarly, distinguishing time-sensitive, e.g., crisis, communications from those that are time agnostic, e.g., background, education, and public awareness information, are crucial for either to have the desired impact. General guidelines and thinking/talking points to enable scientists to work with their counterparts in communications were developed to enable the development of plans to increase food safety through effective communications regarding potential health and financial threats that can result from mycotoxin contamination.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Torres, A. M., S. A. Palacios, N. Yerkovich, J. M. Palazzini, P. Battilani, J. F. Leslie, A. F. Logrieco & S. N. Chulze. 2019. Fusarium head blight and mycotoxins in wheat: Prevention and control strategies across the food chain. World Mycotoxin Journal 12: 333-355. DOI: org/10.3920/wmj2019.2438.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Fumero, M. V., M. Sulyok, M. L. Ramirez, J. F. Leslie & S. N. Chulze. 2020. Effects of water activity and temperature on fusaric and fusarinolic acid production by Fusarium tempera-tum. Food Control 114: 107263. DOI: org/10.1016/j.foodcont.2020.107263
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Fumero, M. V., A. Villani, A. Susca, M. Haidukowski, M. T. Cimmarusti, C. Toomajian, J. F. Leslie, S. N. Chulze, & A. Moretti. 2020. Fumonisin and beauvericin chemotypes and genotypes of the sister species Fusarium subglutinans and Fusarium temperatum. Applied and Environmental Microbiology 86: e00133-20. DOI: org/10.1128/AEM.00133-20.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Leslie, J. F. & J. B. Morris. 2020. Talking about mycotoxins. Frontiers in Sustainable Food Production:Agro-Food Safety 3: 109. DOI: 10.3389/fsufs.2019.00109.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Scientists working with fungi, mycotoxins and the substrates that they contaminate. Changes/Problems:Kevin McCluskey, Curator of the Fungal Genetics Stock Center left at the end of 2018 to accept a job in private industry. A new curator for the collection will be hired during the coming year. What opportunities for training and professional development has the project provided?37 participants attended the 20th annual Fusarium Laboratory Workshop in Manhattan in June 2019 How have the results been disseminated to communities of interest?Fusarium Laboratory Workshop presentations Presentations at professional meetings and publications in professional journals What do you plan to do during the next reporting period to accomplish the goals?Continue in progress research and publish relevant manuscripts. Have begun working with Australian collaborator to revise the 1st edition of The Fusarium Laboratory Manual. This volume is still in print and selling approximately 100 copies per year. Dr. Ensley plans to establish a diagnostic lab for mycotoxins in the College of Veterinary Medicine that should be operational for the next five year phase of this project.

Impacts
What was accomplished under these goals? K-State and the University of Nebraska-Lincoln are the two institutions primarily responsible for mycotoxin work in the USAID-sponsored Feed the Future (FTF) Innovation Lab for the Reduction of Post-Harvest Losses. These efforts have resulted in buy-ins to the project from USAID missions in Kabul, Afghanistan ($1.2 million), Khatmandu, Nepal ($1.2 million), and Tegucigalpa, Honduras ($600,000). Health concerns were of critical importance in all three countries, with economic concerns about exports also important in Afghanistan. The work in Afghanistan is finished, and joint publications are in preparation. Work on raisins and other dried fruits has been in collaboration with BOKU at the University of Vienna (Austria) and with the CNR Institute for the Science of Food Production in Bari, Italy. The work in Nepal was designed to overlap with and extend the work of the FTF Nutrition Innovation Lab to look for correlations between agriculture and the health of pregnant women and infants. Approximately 95% of the women in the Banke district in Nepal are positive for aflatoxin in the blood. Stunting amongst children in this district exceeds 25%. A preliminary survey suggests a potential problem with aflatoxin in chilies and soy nuggets in addition to expected problems in peanuts and maize. A lab was established at the Nepal Academy of Sciences and staff for the lab have been to Nebraska for training. Initial sample analysis is complete with peanuts and maize having the highest levels of aflatoxin contamination, followed by intermediate levels of contamination for dried chilies and soy nuggets, and relatively little aflatoxin contamination in either rice or wheat-based weaning foods. In Honduras the focus was on maize in the western highlands where childhood stunting again exceeded 25% of the population. A testing lab was established at Zamorano University. Maize was the only crop surveyed. Samples were assayed for both aflatoxin and fumonisin, with fumonisin appearing to be the more frequent contaminant. Dr. Steve Ensley is in the process of establishing a mycotoxin analysis lab at K-State. The lab will focus initially on animal feeds. In preliminary studies this year, multiple samples had 10 ppm deoxynivalenol, and others had fumonisins at > 100 ppm. Plans for the coming year are to increase the capacity of the lab and its analytic capabilities. As part of the European Horizon 2020 Research program, a series of roundtable discussion focused on future research areas in mycotoxicology were held at a meeting in Ghent, Belgium. Gary Munkvold from Iowa State also participated in the discussions and is one of 31 co-authors of the resulting paper. A second joint effort was the development and publication of a mycotoxin charter with 17 co-authors to encourage more equitable regulation and increased efforts to address mycotoxin contamination in less-developed countries.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: 1. Leslie, J. F., V. Lattanzio, K. Audenaert, P. Battilani, J. Cary, S. N. Chulze, S. De Saeger, A. Gerardino, P. Karlovsky, Y.-C. Liao, C. M. Maragos, G. Meca, A. Medina, A. Moretti, G. Munkvold, G. Mul�, P. Njobeh, I. Pecorelli, G. Perrone, A. Pietri, J. M. Palazzini, R. H. Proctor, E. S. Rahayu, M. L. Ram�rez, R. Samson. J. Stroka, M. Sulyok, M. Sumarah, C. Waalwijk, Q. Zhang, H. Zhang, & A. Logrieco. 2018. Mycokey round table discussions on future directions in research on chemical detection methods, genetics and biodiversity of mycotoxins. Toxins 10: 109. DOI: 10.3390/toxins10030109.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: 2. Logrieco, A. F., J. D. Miller, M. Eskola, R. Krska, A. Ayalew, R. Bandyopadhyay, P. Battilani, D. Bhatnagar, S. Chulze, S. De Saeger, P. Li, G. Perrone, A. Poapolathep, E. S. Rahayu, G. S. Shephard, F. Stepman, H. Zhang, & J. F. Leslie. 2018. The Mycotox Charter: Increasing awareness of and concerted action for minimizing mycotoxin exposure worldwide. Toxins 10: 149. DOI: 10.3390/toxins10040149.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 3. Vismer, H. F., G. S. Shephard, L. van der Westhuizen, P. Mngqawa, V. Bushula-Njah, & J. F. Leslie. 2019. Mycotoxins produced by Fusarium proliferatum and F. pseudonygamai on maize, sorghum and pearl millet grains in vitro. International Journal of Food Microbiology 296: 31-36. DOI: org/10.1016/j.ijfoodmicro.2019.02.016.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Mohamed Nor, N. M. I., B. Salleh & J. F. Leslie. 2019. Fusarium species from sorghum in Thailand. The Plant Pathology Journal 35: 301-312. DOI: org/10.5423/ppj.oa.03.2019.0049
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: 5. Torres, A. M., S. A. Palacios, N. Yerkovich, J. M. Palazzini, P. Battilani, J. F. Leslie, A. F. Logrieco & S. N. Chulze. 2019. Fusarium head blight and mycotoxins in wheat: Prevention and control strategies across the food chain. World Mycotoxin Journal 12: (in press). DOI: org/10.3920/wmj2019.2438


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:K-State and the University of Nebraska-Lincoln are the two institutions primarily responsible for mycotoxin work in the USAID-sponsored Feed the Future (FTF) Innovation Lab for the Reduction of Post-Harvest Losses. These efforts have resulted in buy-ins to the project from USAID missions in Kabul, Afghanistan ($1.2 million), Khatmandu, Nepal ($1.2 million), and Tegucigalpa, Honduras ($600,000). As a result, we have developed a general process for helping these countries make their own analyses of mycotoxin contamination. Health concerns were of critical importance in all three countries, with economic concerns about exports also important in Afghanistan. The work in Afghanistan is now finished, with two joint publications expected from the research conducted there. Ochratoxin A is limiting the export of raisins and other dried fruits to the European Union. Ergot alkaloids were detected in numerous samples of wheat flour, suggesting that an ergot epidemic may have occurred when samples were being collected. The work in Nepal is designed to overlap with the work of the FTF Nutrition Innovation Lab to look for correlations between agriculture and the health of pregnant women and infants. Approximately 95% of the women in the Banke district in Nepal are positive for aflatoxin in the blood. Stunting amongst children in this district exceeds 25%. The planned survey will look at maize and peanuts, but not everyone consumes these crops. A preliminary survey suggests a potential problem with aflatoxin in chilies and with ochratoxin A in spices. A lab is being established at the Nepal Academy of Sciences and staff for the lab have been to Nebraska for training on equipment similar to what will be used in the lab in Kathmandu. In Honduras the focus will be on maize in the western highlands where childhood stunting again exceeds 25% of the population. Maize will be the only crop surveyed and samples will be assayed for both aflatoxin and fumonisin. The problem might be limited to home-grown corn, although we will test some market samples to ensure that problems are not being imported with grain from outside sources. A lab has been established at Zamorano University. Beauvericins and enniatins are non-ribosomal peptides synthesized by some strains of Fusarium, by an enzyme (enniatin synthetase) that is very similar in both beauvericin-producing and enniatin-producing strains. Structural differences in the β strand of this protein at Asn789-Ala793 and His797-Asp802 distinguish strains that can produce both beauvericin and enniatin from those that produce only ENN. As part of the European Horizon 2020 Research program, a series of roundtable discussion focused on future research areas in mycotoxicology were held at a meeting in Ghent, Belgium. Gary Munkvold from Iowa State also participated in the discussions and is one of 31 co-authors of the resulting paper. A second joint effort was the development and publication of a mycotoxin charter with 17 co-authors to encourage more equitable regulation and increased efforts to address mycotoxin contamination in less-developed countries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Fusarium Laboratory Workshop held at Kansas State University in June 2017 with 52 participants. 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?Publish results of working group discussions and goal-setting, so that the results are available to help policy making and guide the setting of research priorities.. Publish results of Afghanistan surveys to documents the toxin findings from this part of the world, an area for which little information of this sort is available. Collect additional data on contamination of chilis and other foods in Nepal to identify the potential source of aflatoxin that is common in the diet there.

Impacts
What was accomplished under these goals? This project integrates US and international researchers in a common pursuit of increased food security and reduction in contaminated food. Different parts of the world grow, harvest and store crops differently, which can result in different types and levels of fungal contamination of the foodstuff. In Afghanistan, for example, a first step to reducing mycotoxin problems is to institute cleaner drying methods (not just on the ground in the sun). Monitoring different lots for contamination allows the shipper to determine a target country whose regulations will allow the shipment to be received and sold. Rejected shipments can result in significant losses (if the shipment is returned to the home country at the seller's expense) or total losses if the shipment is destroyed. As regulations vary by country, identifying the level of contamination present in a shipment, if any, prior to shipping is critical for reducing losses. Particular mycotoxins are produced by particular fungi. In some cases these fungi are not the same as those found in the United States, especially if the ecosystem in which the agricultural product is grown differs from that in the US. Irrigated wheat is unusual in the United States, but common in South Africa. We found that the fungi responsible for Fusarium Head Blight of wheat, a major disease limiting wheat production, were the same as those found in the major wheat-growing regions of the United States. Our results suggest that problems found in rain-fed wheat in the United States will also be found in irrigated wheat in the country as well. Major research on mycotoxins is occurring outside the United States, especially in Europe. Integrating work with our European colleagues reduces overlap in research efforts and can identify common areas of concern and for future work. Prof. Leslie chaired two working group discussions to identify priorities for research on mycotoxins in the next 3-7 years. These working groups generated a long list of potential research topics as well as a set of areas that the international scientific community considered priorities. Addressing these problem areas will enable the improvement of existing and the development of new strategies to manage and reduce mycotoxin contamination in cereals and in forages. (1) Data collected; manuscript published (2) Chaired international working groups to set priorities for mycotoxin research in the next 3-7 years. (3) Published technical journal article on population genetics of Fusarium isolated from head blight in an unusual ecology - irrigated wheat (wheat usually is grown without irrigation). Published technical journal article on production of enzymes responsible for synthesis of two closely related mycotoxins. Identified potential differences in the enzymes that determine which toxin is synthesized.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 1. Liuzzi, V. C., V. Mirabelli, M. T. Cimmarusti, M. Haidukowski, J. F. Leslie, A. F. Logrieco, R. Caliandro, F. Fanelli, & G. Mul�. 2017. Enniatin and beauvericin biosynthesis in Fusarium species: Production profiles and structural determinant prediction. Toxins 9: 45. DOI: 10.3390/toxins9020045.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 2. Minaar-Ontong, A., L. Herselman, W.-M. Kriel, & J. F. Leslie. 2017. Morphological characterization and trichothecene genotype analysis of a Fusarium Head Blight population in South Africa. European Journal of Plant Pathology 148: 261-269. DOI: 10.1007/s10658-016-1085-5.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audiences for this project are research professionals working in mycotoxins in both post-harvest and pre-harvest scenarios. The efforts made during the reporting period include publications in professional journals, maintaining an international fungal strain reference culture collection, organization of and participation in two laboratory training workshops, and international collaborative research through the USAID-sponsored Feed the Future Innovation Lab for the Reduction of Post-Harvest Losses Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Week long Fusarium Laboratory Workshop was organized at the University of Pretoria in June 2016. 40 participants and seven instructors. Week long Tropical Fusarium Workshop held at the University of Lavras, Brazil, 40 participants and six instructors. Week long conference on mycotoxins in Afghanistan held in New Delhi. 60 participants. Included presentations by local researchers, and outside experts. Conveyed results obtained and suggested future directions for research in Afghanistan through nominal group managed discussions. Participants included ministers and representatives from the Health, Commerce and Agriculture ministries in Afghanistan and commercial exporters and importers of fruits, nuts and wheat. How have the results been disseminated to communities of interest?Professional publications and meeting presentations to inform scientific community of research progress. Two training workshops to help those unfamiliar with fungi in the genus Fusarium and the toxins that it produces begin to be able to work with these fungi. In depth training workshop on Afghanistan results for USAID personnel and in-country stakeholders held in New Delhi. Two presentations at the US Embassy in Kabul for USAID mission and US Embassy personnel and for stakeholders from other international agencies. What do you plan to do during the next reporting period to accomplish the goals?Fusarium Laboratory workshop to be held at Kansas State University. Begin set up for large surveys of mycotoxins in Nepal and Honduras with collaborators in Italy, through an EU-sponsored project, and from the University of Nebraska.

Impacts
What was accomplished under these goals? Objective 1 For nuts from Afghanistan, the in-country analyses suggested that nearly half of the almond and walnut samples, had problems with aflatoxin contamination. These results were not supported by multi-mycotoxin assays conducted at the University of Vienna with LC-MS/MS, which found no aflatoxins in the walnuts and only 6/89 almond samples with levels that would lead to rejection by the EU. More than 50% (26/46) of the pistachio samples were detectably contaminated with aflatoxins, some as high as nearly 3000 μg/kg, and 26% at levels that would lead to rejection by the EU. Aspergillus metabolites were found in 43/46 samples, suggesting that the potential for aflatoxin contamination to increase with storage time is good. Ochratoxin A contamination was detected only at low levels in two walnut (<0.8 μg/kg) and two pistachio (2.5 μg/kg) samples, and in none of the almond samples. In raisins, the results obtained in Afghanistan and at BOKU differed for aflatoxins, with the Afghanistan tests finding nearly 50% of the samples contaminated with aflatoxin while none were positive in the BOKU analyses. The reasons for this discrepancy are not known, but could include the test kit, technical errors in extraction and assay, and non-random splitting of the samples of the samples that were analyzed. Nearly 10% of the samples examined had ochratoxin A levels that would limit exports to the EU, and an additional 14% had some level of contamination. With 99% of the samples carrying evidence for contamination by Aspergillus niger, the likelihood of major ochratoxin contamination problems on exported raisins is high. Wheat is the major staple cereal in Afghanistan with an average Afghan reported to consume ~500 g of wheat daily. Contamination thresholds for food safety estimates are based on common Western diets, and contain much less wheat than is commonly consumed by an average citizen of Afghanistan. The large amount of wheat consumed by Afghans means that even levels of contamination that would be considered "safe" in a Western context may be problematic in an Afghan diet. Thus the frequency at which contaminated samples were detected is probably at least as important a risk factor as the level of contamination present in the samples. Based on the Austrian data for 153 samples, 4% of the samples were positive at some level for aflatoxins, 12% were positive for ochratoxin, 2% were positive for T-2/HT-2, 2% were positive for zearalenone, and 33% were positive for ergot alkaloids. Results from ELISA tests used in Afghanistan to detect T-2 were not consistent and when samples were tested with more sophisticated chemical techniques, T-2/HT-2 was detected at low levels in only four of the 153 samples. Thus, neither zearalenone nor T-2 appears to pose major public health risks in wheat in Afghanistan. Aflatoxin contamination was unexpected, as it is not an important problem on commercial wheat produced in Western countries, and does not usually occur in the field. Poor storage conditions, however, could lead to post-harvest contamination. Ochratoxin A can be a problem in wheat in northern Europe and animals that consume contaminated grain may accumulate the toxin in their muscle. The frequency of ochratoxin A contamination is high enough to be of concern for public safety in Afghanistan, especially since this toxin is associated with kidney failure and this medical condition is a known health problem in the country. Of the 19 contaminated samples, 6 exceeded the European maximum for ochratoxins in cereals for human consumption. Ergot alkaloids were identified in wheat only through the multi-metabolite analyses conducted in Austria. This class of compounds was not a target of the original test protocol, and the few ELISA tests available for these compounds are focused on regulations for animal feeds rather than human food. Ergots were detected in 50/153 samples suggesting that an ergot epidemic had occurred during the crop year from which samples were taken. Ergot is a disease that occurs periodically (depends upon environmental conditions) on grasses and small grains and can be problematic in countries were grain milling options are limited. Objective 2 Nothing to report Objective 3 The Fungal Genetics Stock Center acquired all of the strains used to teach the Fusarium Laboratory Workshop. This acquisition makes the strains more readily available as standards since many had not previously been available from a public collection.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: 1. McCluskey, K., A. Alvarez, R. Bennett, D. Bokati, K. Boundy-Mills, D. Brown, C. T. Bull, M. Coffey, T. Dreaden, C. Duke, G. Dye, E. Ehmke, K. Eversole, K. Fenstermacher, D. Geiser, J. A. Glaeser, S. Greene, L. Gribble, M. P. Griffith, K. Hanser, R. Humber, B. W. Johnson, A. Kermode, M. Krichevsky, M. Laudon, J. Leach, J. F. Leslie, M. May, U. Melcher, D. Nobles, N. R. Fonseca, S. Robinson, M. Ryan, J. Scott, C. Silflow, A. Vidaver, K. M. Webb, J. E. Wertz, S. Yentsch & S. Zehr. 2016. The US culture collection network lays the foundation for progress in preservation of valuable microbial resources. Phytopathology 106: 532-540. DOI: 10.1094/PHYTO-02-16-0074-RVW.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audiences for this project are research professionals working in mycotoxins in both post-harvest and pre-harvest scenarios. The efforts made during the reporting period include publications in professional journals, maintaining an international fungal strain reference culture collection, organization of and participation in two laboratory training workshops, and international collaborative research through the USAID-sponsored Feed the Future Innovation Lab for the Reduction of Post-Harvest Losses Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Week long Fusarium Laboratory Workshop was organized at Kansas State University in June 2015. 32 participants and seven instructors. Week long Tropical Fusarium Workshop held at the University of Vicosa, Vicosa, Brazil, 45 participants and six instructors. How have the results been disseminated to communities of interest?Professional publications and meeting presentations to inform scientific community of research progress. Two training workshops to help those unfamiliar with fungi in the genus Fusarium and the toxins that it produces begin to be able to work with these fungi. What do you plan to do during the next reporting period to accomplish the goals?Fusarium Laboratory workshop to be held at the University of Pretoria, Pretoria, South Africa. Tropical Fusarium workshop to be held at the Narional University of Lavras, Lavras Brazil. Continuing research efforts, especially with data from Afghanistan and further characterization of the molecular genetic organization of the genomes of fungi producing mycotoxins and of the populations of mycotoxin producing fungal species.

Impacts
What was accomplished under these goals? Objective 1 Began work to assess mycotoxin contamination of nuts, dried fruits and wheat in Afghanistan through the USADI mission in Kabul. ordered equipment and sent materials to Afghanistan, hired US staff member to coordinate the project in country. Objective 2 Nothing to report Objective 3 The Fungal Genetics Stock Center maintained holdings of a number of strains critical to the understanding of mycotoxin biosynthesis. Identified and preserved srains from other countries for use in comparisons with strains from the United States. Evaluated number and location of mycotoxin producing gene clusters in several species of Fusarium. Began studies to identify loci that regulate mycotoxin production that are not located in the mycotoxin biosynthetic gene clusters. Developed genetic and physical maps for comparative genomic studies of various mycotixin producing species.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Chiara, M., F. Fanelli, G. Mul�, A. Logrieco, G. Pesole, J. F. Leslie, D. Horner & C. Toomajian. 2015. Genome sequencing of multiple isolates highlights sub-telomeric genomic diversity within Fusarium fujikuroi. Genome Biology and Evolution 7: 3062-3069. DOI: 10.1093/gbe/evv198
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Hernandez-Nopsa, J., G. Daglish, D. Hagstrum, J. F. Leslie, T. Phillips, C. Scoglio, S. Thomas-Sharma, G. Walter & K. Garrett. 2015. Ecological networks in stored grain: Identifying key nodes for emerging pests, pathogens, and mycotoxins in postharvest networks. BioScience 65: 985-1002. DOI: 10.1093/biosci/biv122
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Fanelli, F., M. Chiara, C. Toomajian, G. Pesole, A. F. Logrieco, J. F. Leslie, G. Mul� & D. S. Horner. 2015. Genome sequencing of multiple isolates highlights sub-telomeric genomic diversity within Fusarium fujikuroi. Proceedings of the 13th European Fusarium Seminar: 119.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: McCluskey, K. & J. F. Leslie. 2015. New prospects, progress, and research at the Fungal Genetics Stock Center. Annual Meeting of the American Phytopathological Society (Pasadena, California, USA), Presentation P-796.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Mohammed Nor, N. M. I., W. Yue, C. Toomajian & J. F. Leslie. 2015. Pathogenicity of progeny from a cross between Fusarium fujikuroi and Fusarium proliferatum towards onions. Proceedings of the 13th European Fusarium Seminar: 60.