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United States Department of Agriculture

Agricultural Research Service

Research Project: Protective Endophytes of Maize That Inhibit Fungal Pathogens and Reduce Mycotoxin Contamination

Location: Bacterial Foodborne Pathogens & Mycology Research Unit

2012 Annual Report


1a.Objectives (from AD-416):
The overall goal of this research is to understand how selected ecological groups of symptomless fungal endophytes from maize interact with kernel rotting pathogens and apply this knowledge to reduce disease severity and mycotoxin contamination of the grain. The results obtained through these objectives should produce novel strategies for preventing pathogen related losses in corn productivity and grain quality in a changing global environment. Specific objectives are: Objective 1: Examine the biocontrol potential of Acremonium zeae in providing an effective defense against mycotoxin producing kernel rotting fungi. Objective 2: Discover and characterize metabolites produced by fungal endophytes and pathogens of cereals that support symptomless infection and survival. Objective 3: Characterize fungal endophyte diversity in maize and develop phylogenetic systems to predict the role of novel endophytes in host-pathogen interactions. Objective 4: Determine the production and bioactivity of chitinase modifying proteins (cmp) among common fungal endophytes and pathogens of maize and examine their role in seed pathology.


1b.Approach (from AD-416):
Mycotoxins produced by ear and kernel rotting fungal pathogens of corn are associated with economic losses to maize growers, grain handlers, livestock and poultry producers, and food and feed processors. The safety of mycotoxin-contaminated cereals and cereal products consumed directly by humans as well as mycotoxin residues in animal products is of critical importance to the agri-food industries and regulatory agencies worldwide. No commercial corn hybrid is able to escape aflatoxin or fumonisin contamination when exposed to extensive insect damage, high evening temperatures during kernel filling, or drought. The fungi recorded as symptomless endophytes of corn plants and grain prior to harvest belong to ecologically specialized groups whose interactions potentially influence disease development yet they remain poorly understood. The research proposes to provide new information and strategies for controlling mycotoxin production through: Investigations on the biocontrol potential of the protective endophyte Acremonium zeae; the discovery and analysis of metabolites and proteins that enable endophytes and pathogens to circumvent plant defenses or inhibit competing organisms; an examination of yeast populations in interactions with insects and other fungi; an evaluation of Penicillium subgenus Biverticillium species, known hyperparasites of plant pathogenic fungi; investigations of resistant and susceptible forms of a fungal targeted maize seed chitinase that is presumed to function in protecting seeds from pathogenic fungi; and the development of sorting systems to identify pathogen-specific symptoms of kernel infection and potential mycotoxin contamination. The potential to exploit this poorly understood endophyte-host relationship offers significant promise for protecting corn plants or harvested grain from seedling infection or mycotoxin contamination.


3.Progress Report:
Fungal pathogens that can contaminate corn and other crops with toxins may be able to initiate symptomless infections by producing metabolites which inhibit a protein (Hsp90) essential to a plant immune response. An assay was developed to test purified metabolites for inhibition of this protein. Testing purified fungal metabolites produced by plant pathogens for Hsp90 inhibition is underway. Determining the role of Hsp90 inhibitors in disease progression may allow development of novel pathogen control strategies that reduce toxin levels in food and feed. The role of yeasts as endophytes of maize and other cereals has been largely unexamined, whereas filamentous fungal endophytes have been widely studied. It is unknown if yeasts compete with Fusarium or possibly have the ability to detoxify Fusarium toxins. ARS scientists examined species of the Trichomonascus yeast clade which are known to metabolize complex organic molecules. The 25 known species in this clade were tested for their ability to metabolize T-2 toxin from Fusarium. Yeasts were identified that can modify the T-2 toxin. Three of the species modified T-2 toxin by efficiently attaching a sugar to form a T-2 glucoside. This latter molecule, has been termed a ‘masked’ mycotoxin because it is not detected by standard screening methods. Development of tools to detect and quantify T-2 glucoside in grain has been delayed by the lack of an efficient means to produce or isolate sufficient quantities of these modified toxins. However, T-2 was efficiently converted by the yeasts into T-2 glucoside and will be used to develop rapid analytical methods for detection and measurement of this modified toxin. The discovery of a yeast enzyme system to efficiently produce T-2 glucoside provides an opportunity to determine if conversion to T-2 glucoside represents a detoxification, and will allow us to assess the role of T-2 glucosides in plant pathogen interactions. Putative new species of fungi that can parasitize and kill other fungi, isolated from corn grown in Illinois and surrounding states, were inoculated into milk-stage ears and shown to exclude Fusarium, Stenocarpella, and other kernel rotting and toxin-producing molds from the grain at harvest. Infected seeds from symptomless ears were harvested for experiments that will evaluate the biocontrol potential of these beneficial fungi in germinated seedlings. Detailed descriptions of the appearance of the molds grown in specific laboratory culture are now being documented as the second major step in formally naming the new species. Names are useful for clear communication about toxigenic or biocontrol fungi. Substantial amounts of the DNA sequence data have been entered into a computer database in anticipation of a web-based interface being prepared that will enable accurate identifications of the molds in damaged grain, which is essential in assessing the safety of grain for use in food or feed.


4.Accomplishments
1. Identification of toxins in Stenocarpella rotted corn. Stenocarpella maydis is a fungal pathogen that causes a dry-rot of maize ears and can contaminate grain with neurotoxins that are harmful to livestock. A scientist in the Bacterial Foodborne Pathogens and Mycology Research Unit (BFP), USDA, ARS, National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, collaborated with a scientist at the University of Iowa, Iowa City, IA, to perform chemical investigations of Stenocarpella rotted ears from a field outbreak in Illinois. This research resulted in the isolation of diplodiatoxin and a second set of toxins, chaetoglobosins, as major components. Toxin levels increased ~ 100 fold in grain exposed to a period of post-harvest moist incubation. Contamination of corn ears and stalks with chaetoglobosins could explain reported toxicological effects in livestock grazing harvested fields infested with Stenocarpella. Specific knowledge of the toxins produced by Stenocarpella in corn cultivation is an essential first step in interpreting the mechanism of action by which these toxins affect livestock, poultry or humans, and is critical to the development of practical methods for estimating the levels of these toxins in corn and in assessing potential risk.

2. Discovery of fungal metabolites that inhibit botulism neurotoxin. Fungi that colonize and kill other fungi are potential sources of novel antifungal agents and other compounds useful to agriculture or medicine. A scientist in the Bacterial Foodborne Pathogens and Mycology Research Unit (BFP), USDA, ARS, National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, in collaboration with a University of Iowa scientist in Iowa City, IA, have isolated several antifungal compounds which scientists in the Division of Integrated Toxicology, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, have identified as inhibitors of a neurotoxin causing a life-threatening illness known as botulism. Improperly preserved home-processed foods such as green beans, beets, and corn are a common source of botulism food poisoning. These are the first fungal metabolites reported to inhibit the botulism neurotoxin and can serve as lead compounds for further chemical modifications to improve potency and other pharmacological parameters.

3. Identification of a fungal protein used by Fusarium to cause disease in corn. Fungi known as Fusarium can cause devastating damage to U.S. agriculture through crop loss and by contaminating the food and feed supply with harmful toxins. Plants produce proteins called chitinases that act as a natural defense against fungi. Scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit (BFP), USDA, ARS, National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, previously identified specific plant chitinases that are inactivated by fungal proteins. In the current research, project scientists have isolated and identified one of the fungal proteins responsible for this inactivation. The protein was identified in Fusarium verticillioides, an important fungus that causes corn ear rot and produces toxins that cause adverse health effects. This research has identified a target that can be exploited by chemists, breeders, and genetic engineers to improve disease resistance and reduce mycotoxin contamination of economically important crops.


Review Publications
Naumann, T.A., Price, N.P. 2012. Truncation of class IV chitinases from Arabidopsis by secreted fungal proteases. Molecular Plant Pathology. 13:38-42. DOI: 10.111/j.1364-3703.2012.00805.x.

Samson, R.A., Yilmaz, N., Houbraken, J., Spierenburg, H., Seifert, K.A., Peterson, S.W., Varga, J., Frisvad, J.C. 2011. Phylogeny and nomenclature of the genus Talaromyces and taxa accommodated in Penicillium subgenus Biverticillium. Studies in Mycology. 70(2011):159-183.

Neff, S.A., Lee, S., Asami, Y., Ahn, J., Oh, H., Baltrusaitis, J., Gloer, J.B., Wicklow, D.T. 2012. Aflaquinolones A-G: Secondary metabolites from marine and fungicolous isolates of Aspergillus spp. Journal of Natural Products. 75(3):464-472.

Kurtzman, C.P. 2012. Citeromyces hawaiiensis sp. nov., a new ascosporic yeast associated with Myoporum sandwicense. International Journal of Systematic and Evolutionary Microbiology. 62(pt5):1215-1219.

Samson, R.A., Peterson, S.W., Frisvad, J.C., Varga, J. 2011. New species in Aspergillus section Terrei. Studies in Mycology. 69(1):39-55.

Cardellina, J., Roxas-Duncan, V., Montgomery, V., Eccard, V., Campbell, Y., Hu, X., Khavrutskii, I., Tawa, G.J., Wallqvist, A., Gloer, J.B., Phatak, N.L., Hoeller, U., Soman, A., Joshi, B.K., Hein, S.M., Wicklow, D.T., Smith, L.A. 2012. Fungal bis-naphthopyrones as inhibitors of botulinum neurotoxin serotype A. ACS Medicinal Chemistry Letters. 3(5):387-391.

Dien, B.S., Wicklow, D.T., Singh, V., Moreau, R.A., Moser, J.K., Cotta, M.A. 2012. Influence of Stenocarpella maydis infected corn on the composition of corn kernel and its conversion into ethanol. Cereal Chemistry. 89:15-23.

Naumann, T.A., Wicklow, D.T., Price, N.P. 2011. Identification of a chitinase modifying protein from Fusarium verticillioides: Truncation of a host resistance protein by a fungalysin metalloprotease. Journal of Biological Chemistry. 286(41):35358-35366.

Last Modified: 7/22/2014
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