2007 Annual Report 1a.Objectives (from AD-416) The purpose of the proposed research is to define and control the relationship between economically important plants and three diverse fungal groups that produce mycotoxins on corn and forage grasses. One group includes fungi belonging to the Liseola section of the Fusarium, represented by Fusarium verticillioides (synonym=F. moniliforme), the second includes the black-spored Aspergillus species, and the third group consists of obligate grass endophytes of the family Clavicipitaceae, species of Neotyphodium Glenn, Bacon & Hanlin. There are four objectives: . 1)To decrease the fumonisin mycotoxin content in corn using biocontrol strategies based on the endophytic bacterium Bacillus mojavensis and soil fungi such as Trichoderma koningii;. 2)Define the importance of the fumonisins to corn development and plant or fungus survival, as well as its absorption and translocation in corn;. 3)To develop the use of novel Neotyphodium fungal endophytes in tall fescue for specific reduction of cattle toxins, and enhanced forage persistence measured by nematode resistance; and. 4)To develop an integrated analysis to determine the extent of the mycotoxin ochratoxin A production by isolates of the lesser known but widely prevalent temperate to tropical species Aspergillus niger and A. carbonarius (the Nigri section) that are endophytic to corn kernels, and to determine any co-endophytic interactions with F. verticillioides and/or the fumonisins produced during corn storage. 1b.Approach (from AD-416) Objective 1: Pre-harvest control of fumonisin production will be conducted under controlled conditions of soil moisture, soil inoculum content, over time using corn seedlings and plants of three cultivars that will be inoculated with and without our patented bacterial endophyte, Bacillus mojavensis or Trichoderma koningii, each co-inoculated with strains of Fusarium moniliforme. Fumonisin and fusaric acid, and plant performance will be determined under the above conditions. Objective 2: To address the importance of fumonisin on corn development and virulence factor as well as its translocation by plants, several commercial and experimental corn lines will be screened for tolerance or sensitivity to fumonisin, and identified tolerance and sensitive corn lines will be tested for their ability to translocate fumonisin. All plants will be analyzed for fumonisin content using standard chemical procedures and fungal biomass determined by real-time PCR. The mechanism of corn tolerance will be determined with suppression subtraction hybridization of cDNAs and microarray analyses of sensitive and tolerant lines of corn seedlings. Additionally, a specific approach will target corn genes involved in sphingolipid biosynthesis, such as ASC1. Objective 3: Sterile nematode assays, using two or more nematode species, will be used to determine toxic fractions from the tall fescue endophyte-grass association. The nematode assay will be used both with wild-type and novel endophyte-infected tall fescues. Chemical extracts from infected grasses will be used in the bioassay, and all positive extracts will be separated into specific chemical fractions for chemical identification of the toxin responsible for nematode toxicity. Objective 4: Survey both egg and broiler houses (feed, and litter) for the black-spored Aspergillus niger complex, determine if any of these can produce ochratoxin A on one of several media, including corn, and identify positive isolates to species using molecular techniques. Infect corn seedlings with positive A. niger isolates and determine host-parasite interactions, as well as the effects on toxin accumulation in planta, especially when co-infected with fumonisin producing and non-producing strains of F. verticillioides. 4.Accomplishments Dark Spored Ochratoxin A Producing Aspergillus molds in Corn Used in Poultry Feed (Objective 4). A study designed to determine the distribution and identity of dark spored Aspergillus species in corn indicated that two species from corn were associated with a field case of toxic problem in broilers from this corn used in poultry feed. Preliminary studies have established that one of these dark spored Aspergillus species is a producer of ochratoxin A, a potent mycotoxin with known toxicity to livestock, poultry and humans. This has strong implication to two components of the poultry industry. The level produced by isolates has been shown to alter membrane integrity (tight junction pores) so layer egg shell quality is expected to be affected, along with a variety of other toxic responses broilers. This is the first report of the dark spored species producing the ochratoxin in US corn used for poultry feed. This accomplishment is directly related to 2.1 of Component 2 (Mycotoxin and Plant Toxins) of NP 108—Food Safety and is also related to Problem Statement 2.2 (Crop/Fungal/Toxin Relationships), Problem Statement 2.5 (Biocontrol Technologies), and Problem Statement 2.6 (Toxicity Evaluations and Mechanisms of Action). Origin of corn cultivar insensitivity to the toxin fumonisin is a derived trait (Objective 2). Fumonisin disrupts ceramide synthase activity in corn seedling tissues in a manner similar to that described in animals. We have shown that disruption of ceramide synthase activity results in accumulation of sphingoid bases and their 1-phosphates. A screen of corn, teosinte, and gamagrass lines for ability to germinate on fumonisin found that most were sensitive to fumonisins and either would not germinate or grew very poorly. All gamagrass and teosinte lines and most corn lines were sensitive. Only a few corn lines had significant insensitivity. Sensitivity to fumonisin appears to be the ancestral phenotype, whereas insensitivity is the derived phenotype. While insensitivity may be due to more than one physiological mechanism, differential translocation of fumonisins does appear to play a role. Corn seedlings appear to possess a mechanistic block that selectively translocate primarily fumonisin B1 into foliage, and some corn lines seem to translocate very little fumonisin B1 compared to other lines of corn. These accomplishments are significant regarding potential impact on both science and user groups as they establish a physiological and biochemical basis of endophytic interactions and how this knowledge indicates new and focused directions for plant breeding and reduction of both plant disease and mycotoxin contamination. It is critical that corn breeding programs take into account the endophytic growth of mold such as Fusarium verticillioides and target resistance to infection and fumonisin accumulation and not just disease resistance. This accomplishment is directly related to 2.1 of Component 2 (Mycotoxin and Plant Toxins) of NP 108—Food Safety and is also related to Problem Statement 2.2 (Crop/Fungal/Toxin Relationships), Problem Statement 2.4 (Breeding Resistant Crops), Problem Statement 2.5 (Biocontrol Technologies), and Problem Statement 2.6 (Toxicity Evaluations and Mechanisms of Action). Controlling corn colonization by the toxic fungus Fusarium verticillioides (Objective 3). The frequency of native and introduced populations of the mold Fusarium verticillioides were examined in corn kernels grown under field conditions to analyze sites and mechanisms to target for preventing mycotoxin synthesis in kernels. The introduced F. verticillioides that was used to inoculate corn kernels carried two foreign genes not found in the native toxic F. verticillioides population. The introduced genes served as markers for subsequent isolation and analysis of plant colonization. Results from a multifaceted study indicated that for non-inoculated kernels, 75% tested positive for native Fusarium and 90% of the inoculated kernels tested positive for the F. verticillioides carrying the two foreign genes. There was no evidence of transfer of F. verticillioides carrying the foreign genes from the inoculated to non-inoculated ears, even those wounded following water inoculations. Thus, under normal growing conditions in Georgia, F. verticillioides did not migrate among ears with a high native population of F. verticillioides that exists in ears. Consequently, targeting the elimination of F. verticillioides from corn ears as a control mechanism may be futile. Instead, the mechanisms controlling the switch from symptomless to symptomatic plant colonization. i.e., disease expression might provide a more direct approach for eliminating mycotoxin contamination of foods and feedstuffs that are produced during this change over. This accomplishment is directly related to 2.1 of Component 2 (Mycotoxin and Plant Toxins) of NP 108—Food Safety and is also related to Problem Statement 2.2 (Crop/Fungal/Toxin Relationships), Problem Statement 2.5 (Biocontrol Technologies), and Problem Statement 2.6 (Toxicity Evaluations and Mechanisms of Action). 5.Significant Activities that Support Special Target Populations NONE 6.Technology Transfer Number of active CRADAs and MTAs 1 Number of patent granted 1 Number of web sites managed 1 Number of non-peer reviewed presentations and proceedings 16 Number of newspaper articles and other presentations for non-science audiences 5 Review Publications Bacon, C.W., Hinton, D.M. 2007. Potential for Control of Seedling Blight of Wheat Caused by Fusarium graminearum and Related Species Using the Bacterial Endophyte Bacillus mojavensis. Biocontrol Science and Technology. 17:81-94. Bacon, C.W., Hinton, D.M., Hinton Jr, A. 2006. Growth-inhibiting effects of concentrations of fusaric acid on the growth of bacillus mojavensis and other biocontrol bacillus species. Journal of Applied Microbiology. 100:185-194. Yates, I.E., Sparks, D., Glenn, A.E. 2006. Ear migration in attached and detached sweet corn ears. Journal of the American Society for Horticultural Science. 163:157-161. Bacon, C.W., Hinton, D.M. 2007. Isolation, in planta detection, and uses of endophytic bacteria for plant protection. In: Hurst, C.J., Crawford, R.L., Garland, J.L., Lipson, D.A., Mills, A.L., Stetzenbach, L.D., editors. Manual of Environmental Microbiology. 3rd Edition. Washington, DC:ASM Press. p. 638-651. Hill, N.S., Schwarz, P., Dahleen, L.S., Neate, S., Horsley, R., Glenn, A.E., O Donnell, K. 2006. Elisa analysis for Fusarium in barley: Development of methodology and field assessment. Crop Science. 46:2636-2642. Glenn, A.E. 2007. Mycotoxigenic Fusarium species in animal feed. Animal Feed Science And Technology. 137:213-240.