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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

2013 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:
Acremonium zeae is a beneficial fungus that grows harmlessly in healthy corn plants and has been shown to prevent the spread of kernel-rotting and toxin-producing corn pathogens. Experimental silk-inoculations with A. zeae, performed in successive years using the same commercial hybrid and field location, contributed to a > 60% reduction in the number of kernels infected by F. verticillioides. Acremonium infected seed will be used to evaluate the biocontrol potential of this fungus in germinated seedlings and fungal survival in seeds treated with modern systemic fungicides.

Fungal pathogens and endophytes of corn and other crops 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 and detect the presence of undetermined Hsp90 inhibitors in culture extracts. Determining the role of Hsp90 inhibitors in disease progression may allow development of novel pathogen control strategies.

Cryptococcus flavescens and Cryptococcus luteolus were the predominant fungal colonists (colony forming units) of overwintered maize stalk and cob residues and are being evaluated by ARS National Center for Agricultural Utilization Research chemical engineers for biomass conversion. An ARS scientist in the Crop Bioprotection Research Unit has shown that Cryptococcus flavescens is an effective species for biocontrol of Fusarium head blight. Our discovery of large, natural populations of Cr. flavescens in crop fields provides important information on the ecology of this biocontrol agent.

Four species (21 isolates) of Talaromyces from corn grown in Illinois and surrounding states were evaluated (2nd Yr) for their ability to colonize ears in the milk stage of kernel maturity and exclude naturally occurring fungal endophytes and pathogens (e.g. A. zeae, F. verticillioides, S. maydis). Kernel platings revealed that individual cultures representing two of the Talaromyces species infected each of the kernels with no other fungi being recorded. Talaromyces-infected kernels (1st Yr) exhibited vigorous germination, the fungus spreading into mesocotyl tissues of healthy seedlings. Representative strains of Talaromyces will be examined for their biocontrol potential in protecting corn seedlings from infection by soil-borne fungal pathogens.


4.Accomplishments
1. Discovery of a new fungal protease that degrades plant chitinases. Plant proteins called chitinases have been implicated in many processes, including defense against fungal pathogens, but their biological role is poorly understood. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, previously demonstrated that fungi that cause corn ear rot produce specialized protease proteins that inactivate specific corn chitinases, indicating that the fungi may use this approach to overcome natural plant defenses. Project scientists have also found that another type of fungal protease exists, one that targets the same corn chitinases, but degrades them by recognizing a region that is specific to chitinases from cereals and grasses. In collaboration with an ARS scientist from the Renewable Products Technology Research Unit, Peoria, Illinois, and scientists from the Vavilov Institute of General Genetics, Moscow, Russian Federation, a project scientist identified an antimicrobial peptide from wheat that protects plant chitinases by inhibiting these fungal pathogen proteases. By showing the general nature of this interaction and by revealing its complexity, this research has identified a target that can be exploited by chemists, breeders, and genetic engineers to improve disease resistance of economically important crops.

2. Detection of diplonine neurotoxin produced by Stenocarpella maydis from Midwestern corn. Stenocarpella maydis is a fungal pathogen that causes a dry-rot of corn ears and is associated with ‘diplodiosis’ a neurotoxicosis in cattle grazing harvested corn fields in southern Africa and Argentina. The neurotoxin diplonine was recently isolated by scientists in South Africa from culture extracts of Stenocarpella maydis and shown to induce neurological signs in guinea pigs resembling some of those occurring in cattle and sheep. There have been no reports of Stenocarpella metabolites in corn crop residues. Chemical investigations of Stenocarpella rotted ears from a field outbreak in Illinois were performed by an ARS scientist in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, in collaboration with a University of Iowa scientist in Iowa City, Iowa, leading to the isolation of diplodiatoxin and chaetoglobosins as major components. The results further demonstrated that diplonine was present at very low levels in the rotted ears and in fermentation extracts of Stenocarpella cultures isolated from Midwest corn. These results indicate that diplonine production by Stenocarpella maydis in U.S. corn fields may not pose the same threat to food and feed safety as reported in South Africa.


Review Publications
Naumann, T.A., Wicklow, D.T. 2013. Chitinase modifying proteins from phylogenetically distinct lineages of Brassica pathogens. Physiological and Molecular Plant Pathology. 82:1-9.

Henk, D.A., Eagle, C.E., Brown, K., Van Den Berg, M.A., Dyer, P.S., Peterson, S.W., Fisher, M.C. 2011. Speciation despite globally overlapping distributions in Penicillium chrysogenum: the population genetics of Alexander Fleming’s lucky fungus. Molecular Ecology. 20(20):4288-4301.

Soares, C., Peterson, S.W., Rodrigues, P., Lima, N., Venancio, A. 2012. Three new species of Aspergillus section Flavi isolated from almonds and maize in Portugal. Mycologia. 104(3):682-697.

Dien, B.S., Wicklow, D.T., Singh, V., Moreau, R.A., Winkler-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(1):15-23.

Hughes, S.R., Bang, S.S., Cox, E.J., Schoepke, A., Ochwat, K., Pinkelman, R., Nelson, D., Qureshi, N., Gibbons, W.R., Kurtzman, C.P., Bischoff, K.M., Liu, S., Cote, G.L., Rich, J.O., Jones, M.A., Cedeno, D., Doran-Peterson, J., Riano, N.M. 2013. Automated UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 and selection for microaerophilic growth and ethanol production at elevated temperature on biomass sugars. Journal of Laboratory Automation. 18(4):276-290.

Last Modified: 9/20/2014
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