2007 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.
An application was submitted to EPA for an experimental use permit (EUP) that would allow large-scale field testing of the aflatoxin biocontrol product, afla-guard®, to be carried out on corn grown in Texas. Although many areas of the country can experience problems with aflatoxin contamination of corn in certain years, south Texas has significant aflatoxin in the crop during most years. Therefore, two major corn-producing areas of south Texas were chosen for the study. The EUP was granted in May, 2007, and afla-guard®was manufactured, shipped, and applied to approximately 3000 acres in commercial corn fields. As the crop is harvested, samples of corn are being collected from all treated fields as well as designated control fields. Samples will be evaluated for aflatoxin contamination as well as for determining the impact of the applications on total Aspergillus flavus colonization of corn and incidence of aflatoxin-producing strains of Aspergillus flavus in corn. Although afla-guard® has been registered by the EPA for commercial use on peanuts, its efficacy in corn must be demonstrated before it can be registered for that crop. Obtaining the EUP and carrying out this large-scale study represents significant progress in having a proven biocontrol product registered for use in another aflatoxin-susceptible crop.
Extend the shelf life of afla-guard7: In the prior project an aflatoxin biocontrol product was developed, commercialized, and registered with EPA as a biopesticide called afla-guard7. One of the materials used in the manufacture of afla-guard7 was soybean oil, which was used to suspend spores of the nontoxigenic strain of Aspergillus flavus being used as the active ingredient (ai). The only disadvantage associated with the afla-guard7 formulation was that as the oil became rancid, oil breakdown products were released that were toxic to the ai, which limited its shelf life, particularly when stored at higher temperatures. Therefore, a study was carried out whereby afla-guard7 was prepared with eight different oils representing a range of oxidative stabilities and stored at five different temperatures ranging from 4 to 44¿ C. Samples were analyzed monthly for over a year to determine product stability. It was found that afla-guard7 made with mineral oil greatly improved the shelf life of the product, extending it from
about seven months for afla-guard7 made with soybean oil to over 19 months for afla-guard7 made with mineral oil. This means that unused product from one year can be used in the following year, which is a major benefit to the manufacturer and users. This accomplishment addresses National Program been developed. The method is flexible and can be applied for quantitation of the defensive stilbenoids in different parts of the peanut plant. The method was used in the determination of the peanut kernel responses to fungal challenge under laboratory conditions. Molar extinction coefficients for the major stilbene phytoalexins, arachidin-1 and arachidin-3, were determined for the first time, which enables reliable quantitation of these stilbenoids without the use of labile, commercially unavailable standards. This accomplishment addresses National Program 108 Component 2 (Mycotoxins and Plant Toxins), and addresses Problem Statement 2.1.2, Crop/Fungal/Insect/Toxin Relationships.
Develop an assay to evaluate nontoxigenic strains of Aspergillus flavus: A laboratory peanut seed assay system was developed and optimized in order to quickly evaluate nontoxigenic strains of Aspergillus flavus as potential aflatoxin biocontrol agents. Using this assay, different toxigenic and nontoxigenic strains can be quantified and inoculated onto wounded peanut seeds to determine relative strain competitiveness. Thus, nontoxigenic strains with enhanced abilities to compete with toxigenic strains can be identified for further field evaluation. Use of this assay can save much time in the identification of strains to be used as future biocontrol agents, as the non-competitive strains can be quickly ruled out. This accomplishment addresses National Program 108 Component 2 (Mycotoxins and Plant Toxins), and addresses Problem Statement 2.1.5, Biocontrol Technologies.
Develop methodology to quantify chemical responses of peanut to fungal challenge: A simple and affordable chemical method for simultaneous determination of peanut stilbenoids in thin slices of peanut kernels has been developed. The method is flexible and can be applied for quantitation of the defensive stilbenoids in different parts of the peanut plant. The method was used in the determination of the peanut kernel responses to fungal challenge under laboratory conditions. Molar extinction coefficients for the major stilbene phytoalexins, arachidin-1 and arachidin-3, were determined for the first time, which enables reliable quantitation of these stilbenoids without the use of labile, commercially unavailable standards. This accomplishment addresses National Program 108 Component 2 (Mycotoxins and Plant Toxins), and addresses Problem Statement 2.1.2, Crop/Fungal/Insect/Toxin Relationships.
5.Significant Activities that Support Special Target Populations
|Number of new CRADAs and MTAs||1|
|Number of non-peer reviewed presentations and proceedings||9|
Dorner, J.W., Horn, B.W. 2007. Separate and combined applications of nontoxigenic aspergillus flavus and a. parasiticus for biocontrol of aflatoxin in peanuts
. Mycopathologia. 163:215-223.
Dorner, J.W., Lamb, M.C. 2006. Development and commercial use of afla-guard®, an aflatoxin biocontrol agent. Mycotoxin Research. 21:33-38.
Sobolev, V., Deyrup, S.T., Gloer, J.B. 2006. A new peanut (arachis hypogaea) phytoalexin with stilbene and but-2-enolide moieties. Journal of Agricultural and Food Chemistry. 54:2111-2115
Sobolev, V., Guo, B., Holbrook Jr, C.C., Lynch, R.E. 2007. Interrelationship of Phytoalexin Production and Disease Resistance in Selected Peanut Genotypes. Journal of Agricultural and Food Chemistry. 55:2195-2200.
Sobolev, V. 2007. Simple, fast and cheap cleanup method for quantitation of aflatoxins in important agricultural products by HPLC
. Journal of Agriculture and Food Chemistry. 55:2136-2141.
Whitaker, T.B., Dorner, J.W., Lamb, M.C., Slate, A. 2007. The effect of sorting farmers' stock peanuts by size and color on partitioning aflatoxin into various shelled peanut grade sizes. Peanut Science 32:103-118.
Sobolev, V., Potter, T.L., Horn, B.W. 2006. Prenylated stilbenes from peanut root mucigel. Phytochemical Analysis. 17:312-322.
Horn, B.W. 2006. Relationship between soil densities of Aspergillus species and colonization of wounded peanut seeds. Canadian Journal of Microbiology. 52:951-960.