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
Recombinant HSP90 protein has been expressed in Escherichia coli. The protein was purified and is ready for use in the inhibitor screens. A series of pure compounds, isolated from phytopathogenic fungi, have been obtained for use in initial screens. Representative yeasts were deposited in the Agricultural Research Service (ARS) Culture Collection from past studies on the microbiology of corn, wheat, and rice. These isolates (213 strains) have been retrieved from the culture collection and are being identified from gene sequence analysis to provide background on the species typically present on these field crops. Citreoviridin production by Aspergillus terreus was confirmed and four other species in Aspergillus section terrei are documented as citreoviridin producers. Citreoviridin production by three new Penicillium species was also discovered. The biodiversity of Penicillium subgenus Biverticillium is much greater than current estimates. Approximately 20 undescribed species have been discovered in this subgenus using phylogenetic analysis of DNA sequences. Those new species can now be described. A chitinase modifying protein (cmp) has been purified from a fungal pathogen of corn and amino-acid sequence information is being collected to identify this protease. Analysis of the reaction mechanism has determined that it is an endoprotease that cleaves maize ChitA at a specific peptide bond. Also, recombinant class IV chitinases, produced from cDNAs from a dicotyledenous plant, have been purified and are being tested as cmp substrates.
1. Identification of a general method used by fungi to overcome plant defenses. Fungi 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. Agricultural Research Service (ARS) scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit in Peoria, IL previously demonstrated that fungi that cause corn ear rot produce proteins that inactivate corn chitinase, indicating that the fungi may use this approach to overcome natural plant defenses. In the current research, project scientists discovered that these fungi can also inactivate similar plant defense proteins from other plants. In addition, it was discovered that a fungal pathogen of crops in the cabbage family (broccoli, cabbage, cauliflower, canola, radish) also makes a protein that inactivates plant chitinases. These results indicate that fungal inactivation of plant chitinases is a general method used by fungi to overcome plant defenses. By showing the general nature of this interaction, this research has identified a target that can be exploited by chemists, breeders, and genetic engineers to improve disease resistance of economically important crops.
Tallada, J.G., Wicklow, D.T., Pearson, T.C., Armstrong, P.R. 2011. Detection of fungus-infected corn kernels using near-infrared reflectance spectroscopy and color imaging. Transactions of the ASABE. 54(3): 1151-1158.