2008 Annual Report 1a.Objectives (from AD-416) Classify and optically sort maize grain infested with common species of kernel rotting fungi and contaminated with mycotoxins. Identify and characterize gene products from Fusarium verticillioides essential to fungal competitive success that respond to chemicals from microbial constituents growing on or in corn. Discover and characterize proteins from protective endophytes of maize and novel mycoparasitic fungi that are capable of inhibiting growth of Aspergillus flavus and F. verticillioides. Provide information and germplasm that will be used by private seed companies to create corn hybrids that resist accumulation of aflatoxin. Investigate protein expression in ripening maize grains challenged by protective endophytes versus destructive pathogens. Examine the ecological role of Acremonium zeae and other protective endophytes of maize in providing effective defenses against mycotoxin producing kernel rotting fungi. Discover and characterize novel antifungal metabolites produced by mycoparasites and fungal endophytes of maize and other cereals with activity against Aspergillus and Fusarium. 1b.Approach (from AD-416) Train neural networks to classify corn kernel disease symptom expression in "resistant" vs. "susceptible" white or yellow maize lines infected with by different species of fungi; identify filters for detecting and rejecting mycotoxin contaminated maize grains in commercially available high speed optical sorters; identify genes that respond to maize or are required for growth on maize via analysis of the Fusarium verticillioides Expressed Sequence Tag (EST) database and F. verticillioides genes that respond to competition via microarray analysis; obtain protein sequence information on promising mycoparasite antifungal protein candidates via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis and use this information to identify and clone the corresponding genes; characterize the impact of stress on plant disease resistance gene products (PDRGPs) and F. verticillioides growth in developing maize ears; evaluate PDRGPs and F. verticillioides ear rot reactions in agronomically characterized germplasm for use as markers in ear rot resistance breeding programs; investigate the biocontrol potential of Acremonium zeae in maize cultivation; evaluate maize endophyte metabolites for their antifungal activities against Aspergillus flavus and Fusarium verticillioides as well as other biological effects relevant to agriculture or medicine. 3.Progress Report Our research has demonstrated that high speed optical sorting is a viable method to remove moldy corn kernels. Near infrared reflectance wavelength pairs were identified resulting in significant reductions in aflatoxin and fumonisin in commercial seed samples. Work continues to elucidate the interaction and impact of two naturally occurring fungal endophytes of maize. Acremonium zeae is a protective endophyte with antifungal activity against other fungi. Pyrrocidines A and B are commonly found in healthy grain from milder climates and are active in bioassays against major stalk and ear rot fungi but are inactive against protective endophytes and mycoparasites. Under stress conditions, such as heat or drought, Fusarium verticillioides changes from a non-symptomatic endophyte to a pathogen and produces fumonisin. Using fluorescent protein tagged F. verticillioides strains and diverse maize germplasm, we are evaluating the role of stresses and plant genetics on fungal colonization and fumonisin formation. Proteins from both fungus and plant origin were identified that are involved in disease resistance in maize. Loss of resistance is due to direct modification of the plant protein by the fungal protein. Additional novel antifungal metabolites from mycoparasites and maize endophytes were evaluated for efficacy against Aspergillus flavus and F. verticillioides thereby adding to the structure-function relationship database. Microarray analyses of F. verticillioides exposed to metabolites from fungi often found contaminating maize identified a number of up-regulated and down-regulated genes. A prioritized list for further analysis was developed. This research addresses NP 108, Component 2. 4.Accomplishments 1. FUSARIUM GENE PRODUCTS FOR GROWTH OR MAIZE DEFENSE. Microarray analysis of Fusarium verticillioides exposed to metabolites synthesized by fungi often found contaminating maize has identified a number of up-regulated and down-regulated genes. We are in the process of analyzing the data to create a prioritized list of genes and proteins for further analysis. We have continued our examination of the six KP4-like genes in F. verticillioides by identifying three KP4 homologs in Epichloe festucae, as well as two in the moss Physcomitrella patens. The genomic organization of the three E. festucae KP4 genes is identical to Fusarium and raises the possibility that they were attained by horizontal gene transfer. Phylogenetic analysis suggest that the development of the KP4 dimers evolved on two occasions; once in Fusarium and again in Phaeosphaeria nodorum. Expression analysis in F. verticillioides of two KP4 genes by microarrays has revealed that at least one is highly differentially regulated. Disruption vectors were created to examine function of all of the KP4 genes in F. verticillioides. The goal of this research is to identify F. verticillioides genes that augment maize plant defenses. This research contributes to NP 108, Component 2, Problem Area 1.4. 2. ACREMONIUM ZEAE AND PYRROCIDINES IN ENDOPHYTE DEFENSE OF MAIZE. Acremonium zeae produces pyrrocidines A and B, polyketide-amino acid-derived antibiotics. Pyrrocidine A exhibited potent activity in bioassays against several major stalk and ear rot pathogens of corn. Whereas, protective fungal endophytes and mycoparasites that grow asymptomatically within healthy corn tissues showed little sensitivity. Pyrrocidine A also exhibited potent activity against Clavibacter michiganense subsp. Nebraskense, causal agent of Goss’s bacterial wilt of corn, as well as bacteria from corn being evaluated as biocontrol agents. An evaluation of a random sampling of A. zeae isolates accessioned by the Agricultural Research Service (ARS) and Centraalbureau voor Schimmelcultures (CBS) Culture Collections from 1969-1992 revealed that pyrrocidines were produced by 27 percent of the isolates from maize grown in regions with milder climates (e.g., Illinois, Nebraska, and Germany), and 83 percent of the isolates from maize grown in warmer regions (e.g., North Carolina, Georgia, Iran, and India). In regions where maize is more vulnerable to pathogen attack following the damaging effects of drought and temperature stress, selection may favor A. zeae endophytes that produce pyrrocidines as acquired chemical defenses. This research contributes to NP 108, Component 2, Problem Area 1.4. 3. DETECTING AND SORTING MYCOTOXINS CONTAMINATED GRAINS. Near infrared reflectance spectra (500-1700 nm) were analyzed to identify single whole white corn kernels contaminated with aflatoxin and fumonisin. Kernels were visually examined and grouped into four symptom categories: asymptomatic, showing 25% to 50% discoloration, showing over 75% discoloration, and discolored bright green yellow fluorescence (BGYF) kernels. Friable kernels and fragments were not included in this study as they are usually removed by existing cleaning equipment at grain elevators. Spectra were acquired on both the germ and endosperm sides of each kernel. Aflatoxin B1 or total fumonisins (B1, B2, and B3) were measured with a fluorometer after extracts were purified with immunoaffinity columns (Aflatest or Fumonitest, Vicam, Watertown, MA). The aflatoxin or fumonisin level of each five-kernel group then was assigned to each individual kernel from that group. Kernels were analyzed in groups to reduce cost and analysis time. For high speed sorting operations, whole spectra cannot be acquired at throughput rates that are economically feasible. Discriminate analysis was used to select the optimal pair of wavelengths to identify kernels containing mycotoxins. It was found that using the wavelength pair of 500nm and 1200nm, approximately 87% and 93% of kernels having high levels of aflatoxin (>100ppb) and high levels of fumonisin (>40ppm), respectively, were correctly classified. Additionally, approximately 96% of the kernels having low levels of aflatoxin (<10ppb) and fumonisn (<2 ppm) were correctly classified as uncontaminated. Kernels having minor symptoms (25% to 50% discolorations) had lower classification accuracies (80%), than those with discolorations covering more than 75% of the kernel (88%), or discolored BGYF kernels (91%). This research contributes to NP108, Component 2, Problem Area 1.3. 4. NOVEL ANTIFUNGAL METABOLITES FROM MYCOPARASITES AND MAIZE ENDOPHYTES. There is an urgent need for new sources of antifungal agents. Fungi that parasitize and kill other fungi as well as protective fungal endophytes that inhabit maize plants are excellent potential sources of novel antifungals, as well as new compounds that might have other useful effects. Using two important mycotoxin-producers, Aspergillus flavus and Fusarium verticillioides, as targets, a University of Iowa scientist in collaboration with an Agricultural Research Service (ARS) scientist in Peoria, Illinois, have isolated and identified numerous antifungal metabolites that show activity against the target organisms, and/or effects on agriculturally important insect pests or in anticancer screens. Versicolamide B, produced by Aspergillus versicolor, was reported as new metabolite with rare stereochemistry belonging to a family of alkaloids known for its myriad of biological activities, while Botryolides A-D, produced by Chaetomium piluliferum, were reported as new fungal metabolites which are closely related to decarestrictines, a class of metabolites found to have a wide range of bioactivities. These ongoing studies are contributing to a growing database that will be useful in evaluating the biocontrol potential of fungal endophytes in preharvest maize, structure-function relationships and potential cellular targets in Aspergillus and Fusarium. The research continues to be supported in part with a grant from the National Institutes of Health. Other aspects of cooperative research between these two laboratories are supported by a grant from the National Science Foundation. This research contributes to NP 108, Component 2, Problem Area 1.4. 5. MAIZE PROTEINS EXPRESSED DURING ENDOPHYTE VS. PATHOGEN INFECTION. We implemented a proteomics strategy to identify a disease resistance gene product (PDGRP) whose activity is substantially reduced in developing seeds of ears inoculated with a known ear-rot pathogen. We have determined that the loss of PDGRP activty is due to direct modification of this protein by a fungal protein. We have purified both the fungal protein and corn PDGRP and can reproduce the interaction in vitro. We also analyzed more than 20 lines of corn and found that the PDGRP is present in all cases. Polymerase chain reaction (PCR) primers that specifically recognized the FUM8 gene, and green fluorescent protein (GFP) used to tag the FUM8 gene, were developed and used to track Fusarium verticillioides in plants of several corn lines and hybrids growing under ideal and under stress conditions. Fungal colonization was detected in stalk, shank, cob, tassel, silk, and kernel tissue. This research seeks to identify how Fusarium moves through a developing corn plant and determine what, if any, influence host genotype has on fungal colonization and fumonisin formation. This research contributes to NP 108, Component 2, Problem Area 1.4. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings 5 Review Publications Wicklow, D.T., Horn, B.W. 2007. Association between vegetative compatibility and aflatoxin production by Aspergillus species during intraspecific competition. Mycoscience. 48:267-273. Wicklow, D.T., Mcalpin, C.E., Yeoh, Q. 2007. Diversity of Aspergillus oryzae genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia. Mycoscience. 48(6):373-380. Greshock, T.J., Grubbs, A.W., Jiao, P., Wicklow, D.T., Gloer, J.B., Williams, R.M. 2008. Isolation, structure elucidation, and biomimetic total synthesis of versicolamide B and the isolation of antipodal (-)-stephacidin A and (+)-notoamide B from Aspergillus versicolor. Angewandte Chemie. 47(19):3573-3577. Sy, A.A., Swenson, D.C., Gloer, J.B., Wicklow, D.T. 2007. Botryolides A-E, Decarestrictine Analogues from a Fungicolous Botryotrichum sp. (NRRL 38180). Journal of Natural Products. 71(3):415-419. Poling, S.M., Wicklow, D.T., Rogers, K., Gloer, J. 2008. Acremonium zeae, a protective endophyte of maize, produces dihydroresorcylide and 7-hydroxydihydroresorcylides. Journal of Agricultural and Food Chemistry. 56(9):3006-3009. Available: http://pubs.acs.org/cgi-bin/abstract.cgi/jafcau/2008/56/i09/abs/jf073274f.html.