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United States Department of Agriculture

Agricultural Research Service

2009 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.

3.Progress Report
The second year of studies under an EPA-approved two-year Experimental Use Permit (EUP) to determine the efficacy of the aflatoxin biocontrol product, afla-guard®, in corn was completed. Through the cooperation of several Texas corn producers and the manufacturer of afla-guard, Circle One Global, Inc., approximately 3000 acres in two major corn-producing areas of south Texas were treated with afla-guard. Four hundred thirty-eight corn samples were collected from 58 fields and evaluated for aflatoxin contamination and A. flavus colonization. Results showed that applications of afla-guard reduced mean aflatoxin levels in corn by 88%. Laboratory and field plot studies were again carried out in the evaluation of several nontoxigenic strains of A. flavus for their competitiveness against toxigenic strains and control of aflatoxin contamination. Laboratory studies utilized a unique method that involves equilibration of seeds at the appropriate water activity and inoculation of those seeds with specific quantities of various toxigenic and nontoxigenic strains. Relative competitiveness of different strains was evaluated and best performing nontoxigenic strains were further evaluated in peanut field plots in which optimum conditions for aflatoxin contamination could be imposed. Long-term effects of application of nontoxigenic strains to peanut and corn plots was evaluated with respect to the composition of A. flavus soil populations. Important questions that need to be answered from this research are how often and for how long do applications of nontoxigenic strains need to be made to maintain control of aflatoxin contamination. The need for discovery of new antifungal, antimicrobial, and anticancer agents that possess low mammalian and low environmental toxicity is recognized by the scientific community as well as commercial institutions. It is believed that some of the best new chemistries, which often are difficult to synthesize, can be obtained from natural sources. In a study of mature peanut seed response to fungal challenge it was revealed that numerous stilbenoid compounds of unusual structure were actively produced by peanuts at different periods of incubation. The structures of four new isolated compounds were determined by modern spectroscopic analysis. At present, the compounds are being investigated for antifungal activity against various plant pathogens, including Colletotrichum acutatum, C. fragariae, C. gloeosporioides, Phomopsis viticola, P. obscurans, Botrytis cinerea, and Fusarium oxysporum. In preliminary research some compounds demonstrated inhibitory activity against selected species at very low concentrations. The new compounds are being tested also in insect studies against mosquitoes, turnip and azalea lace bugs, and fire ants. Cytotoxicity of the new compounds against important cancer cells and their antioxidant activities are also being investigated.

1. Aflatoxin biocontrol technology gains EPA approval for use on corn. Large-scale studies to test the efficacy of afla-guard for control of aflatoxin contamination in corn were conducted under an EPA-approved Experimental Use Permit. Results of the two-year EUP were submitted to EPA, which approved it for use on corn beginning with the 2009 crop. This biocontrol technology is now labeled for use on both peanuts and corn.

2. Sexual reproduction in aflatoxin-producing fungi. The fungi Aspergillus flavus and A. parasiticus infect a wide variety of crops, including corn, peanuts, cottonseed and tree nuts, and contaminate these food sources with aflatoxin, the most potent naturally occurring carcinogen known. The cost of regulating aflatoxin in commodities for domestic use and foreign export in the United States is substantial. Both fungal species were previously considered strictly nonsexual and clonal in their ability to reproduce and disperse. Nevertheless, field populations of these fungi are extremely diverse genetically and the origin of this diversity in the absence of sexuality was always a mystery. In collaborative research with North Carolina State University, the sexual stage for both A. flavus and A. parasiticus was discovered using both classical and molecular research approaches. Furthermore, the research demonstrated that sexual reproduction involved genetic recombination leading to variation in the offspring. The discovery of sexuality in aflatoxin-producing fungi will result in a reassessment of all aspects of their biology and will allow scientists to perform genetic crosses to understand the heritability of the aflatoxin genes in these economically important fungi.

3. New phytoalexins isolated from peanuts. Four new antifungal compounds have been isolated from peanut seeds challenged by a fungal strain, along with two known compounds that have not been previously reported in peanuts. The structures of these new putative phytoalexins were determined. Together with other known peanut stilbenoids that were also produced in the challenged kernels, these new compounds may play a defensive role against invasive fungi.

6.Technology Transfer

Number of Other Technology Transfer1

Review Publications
Horn, B.W., Ramirez-Prado, J.H., Carbone, I. 2009. Sexual reproduction and recombination in the aflatoxin-producing fungus Aspergillus parasiticus. Fungal Genetics and Biology. 46(2):169-175.

Sobolev, V.S., Neff, S.A., Gloer, J.B. 2008. New Stilbenoids from Peanut (Arachis hypogaea) Seeds Challenged by an Aspergillus caelatus Strain. Journal of Agricultural and Food Chemistry. 57(1):62-68.

Horn, B.W., Dorner, J.W. 2009. Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations. Biocontrol Science and Technology. 19(30:249-262.

Horn, B.W. 2009. The sexual state of Aspergillus parasiticus. Mycologia. 101(2:275-280.

Sheppard, G.S., Berthiller, F., Dorner, J.W., Krska, R., Lombaert, G.A., Malone, B., Maragos, C.M., Sabino, M., Trucksess, M., Whitaker, T.B. 2009. Developments in mycotoxin analysis: an update for 2007-2008. World Mycotoxin Journal 2:3-21. .

Chang, P.-K., Horn, B.W., Dorner, J.W. 2009. Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus. Fungal Genetics and Biology. 46:176-182.

Dorner, J.W. 2009. Development of Biocontrol Technology to Manage Aflatoxin Contamination in Peanuts. Peanut Science. v.36 pp 60-67.

Dorner, J.W. 2008. Relationship Between Kernel Moisture Content and Water Activity in Different Maturity Stages of Peanut. 2008. Peanut Science. v 35 pp 77-80.

Dorner, J.W. 2009. Biological Control of Aflatoxin Contamination in Corn using a Nontoxigenic Strain of Aspergillus flavus. Journal of Food Protection. 2009. V.72 pp 801-804.

Last Modified: 11/28/2015
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