2008 Annual Report
1a.Objectives (from AD-416)
1. Refine aflatoxin biocontrol technology for peanuts and develop an effective system for achieving biological control of aflatoxins in corn, an important crop grown in rotation with peanuts. 2. Determine characteristics of soil populations important for invasion of peanut seeds by aflatoxigenic fungi and evaluate the competitiveness of nontoxigenic biocontrol strains of A. flavus. 3. Determine the chemical barriers of peanut to fungal challenge, particularly challenge by A. flavus. Investigate the basis for greater resistance to A. flavus invasion and aflatoxin contamination possessed by certain peanut genotypes for possible exploitation in breeding programs.
4. Conduct the necessary laboratory and field trials required by the EPA to extend the use of Aflaguard to other crops susceptible to aflatoxin, such as corn.
1b.Approach (from AD-416)
Experiments to extend the shelf life of afla-guard(r) will be conducted by producing afla-guard(r) with a variety of oils covering a range of oxidative stabilities. Samples will be placed in long-term storage at 4 degrees, 23 degrees, 30 degrees, 37 degrees, and 44 degrees C and tested once a month to determine the survival and viability of conidia on the coated barley. A multi-year (at least three) study will be conducted to determine the possibility of achieving biological control of aflatoxin contamination of corn. The field tests will include two plantings (3-4 weeks apart) of four treatments in a randomized complete block design with eight replications. Corn will be ground in a Romer subsampling mill, and the quantity and toxigenicity of A. flavus in the corn will be determined. Aflatoxins will be quantified in the same samples.
Native fungal populations in 20 different soils will be quantified and species will be identified either directly on the dilution plates or by subculturing to Czapek agar slants. Peanut seeds will be aseptically wounded and inoculated with 7.0 mg of soil paste using a small spatula. Forty seeds will be inoculated with each soil and incubated 14 d at 37 C. Twenty-four uninoculated wounded seeds will serve as controls in each experiment. A. flavus and A. parasiticus sporulating on seeds will be identified by subculturing to Czapek agar slants. In a related series of experiments, nontoxigenic biocontrol strains (conidial-color mutant A. parasiticus NRRL 21369 and a nitrate-nonutilizing mutant of A. flavus NRRL 21882) will be added to soils at different concentrations to examine their interactions with native aflatoxin-producing populations. Aflatoxin analyses of individual seeds will be performed by extracting overnight in methanol and quantifying with high performance liquid chromatography.
A series of experiments will be conducted to.
1)isolate, identify, and quantify chemicals produced in peanuts in response to fungal invasion;.
2)characterize the chemical response of peanuts representing a range of pod/kernel maturity to fungal challenge;.
3)characterize the chemical responses of peanuts representing a genotypic range of recognized differences in susceptibility to A. flavus invasion and aflatoxin contamination;.
4)characterize peanut wax composition and evaluate different genotypes for peanut wax content and composition.
The first year of studies under an Environmental Protection Agency (EPA) approved two-year experimental use permit to determine the efficacy of the aflatoxin biocontrol product, afla-guard®, in corn was completed. Approximately 3000 acres in two major corn-producing areas of south Texas were inoculated with afla-guard, and 450 samples were evaluated for aflatoxin contamination and A. flavus colonization. Results showed that applications of afla-guard reduced mean aflatoxin levels in corn by 85%. The second year of the study is underway with an additional 3000 acres of corn having been treated. Corn is currently being harvested and samples collected for aflatoxin analyses. Data from the study is necessary to obtain Environmental Protection Agency approval for use of the biopesticide to control aflatoxin in corn. In a study of peanut kernel response to fungal invasion it was determined that phenylpropanoid compounds that encompass a range of structural classes play a role in peanut resistance to 15 important soil fungal strains in the genus Aspergillus. Stilbene derivatives and phenolic acids were the major components produced by fungal-challenged peanuts. There was a significant difference in phytoalexin production elicited by some fungal strains. Production of stilbenoids and free and bound phenolic acids by the peanut plant at early stages of growth was also investigated, demonstrating a role for phenolic acids as defensive compounds against fungi. A new spermidine derivative was isolated from peanut flowers with structure elucidation based on spectroscopic evidence. Another spermidine conjugate as well as four flavonoid conjugates were determined in peanut flowers for the first time. This research addresses National Program 108 (Food Safety), Component 2 (Mycotoxins and Plant Toxins), Problem Statements 2.1.5 (Biocontrol Technologies) and 2.1.2 (Crop/Fungal/Insect/Toxin Relationships).
A laboratory assay was developed to evaluate nontoxigenic strains of Aspergillus flavus as potential biocontrol agents. The assay system, which involves the inoculation of viable, wounded peanuts, has shown that nontoxigenic A. flavus is more competitive than nontoxigenic A. parasiticus in controlling aflatoxins and that the effectiveness of both species depends upon the density and toxigenicity of native soil populations of aflatoxin-producing fungi. Furthermore, evaluation of eight nontoxigenic A. flavus strains using the peanut seed assay suggests that several strains may be more effective at preventing aflatoxins than the current strain comprising afla-guard®. The potential impact of this accomplishment is the development of a more effective aflatoxin biocontrol product. This accomplishment addresses National Program 108 (Food Safety), Component 2 (Mycotoxins and Plant Toxins), Problem Statement 2.1.5 (Biocontrol Technologies).
5.Significant Activities that Support Special Target Populations
Dorner, J.W. 2008. Management and Prevention of Mycotoxins in Peanuts. Journal of Food Additives & Contaminants. 25:203-208.
Horn, B.W. 2007. Biodiversity of Aspergillus section Flavi in the United States. Journal of Food Additives & Contaminants. 24(10):1088-1101.
Ramirez-Prado, J.H., Moore, G.G., Horn, B.W., Carbone, I. 2008. Characterization and Population Analysis of the Mating-Type Genes in Aspergillus flavus and A. parasiticus. Fungal Genetics and Biology. 45(9):1292-1299.
Carbone, I., Jakobek, J.L., Ramirez-Prado, J.H., Horn, B.W. 2007. RECOMBINATION, BALANCING SELECTION AND ADAPTIVE EVOLUTION IN THE AFLATOXIN GENE CLUSTER OF ASPERGILLUS PARASITICUS. Molecular Ecology. 16(20):4401-4417.
Carbone, I., Ramirez-Prado, J.H., Jakobek, J.L., Horn, B.W. 2007. Gene duplication, modularity and adaptation in the evolution of the aflatoxin gene cluster. Journal of Evolutionary Biology 7:111.