2010 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.
Nontoxigenic strains of Aspergillus flavus were further evaluated in laboratory and field plot studies. The laboratory assay involved the co-inoculation of viable, artificially wounded peanut seeds with mixtures of nontoxigenic and aflatoxin-producing strains. Eight nontoxigenic strains (including the strain present in afla-guard®) that differed genetically in their inability to produce aflatoxins were paired with eight genetically different aflatoxin-producing strains in all combinations. The second of two sets of replicate experiments was completed and the data were analyzed statistically. Four of the most promising nontoxigenic strains for reducing aflatoxins in the laboratory assay (based on the first set of laboratory experiments) were tested for a third year in the environmental control plots in which peanut plants are subjected to elevated soil temperatures and drought stress to increase aflatoxin contamination. Due to the extreme variability in aflatoxin levels from year to year, the nontoxigenic strains could not be adequately evaluated in the plots and will require further field testing.
Selected peanut cultivars that differed in their resistance to A. flavus infection were challenged by fungi from different genera. Research involved the detection, isolation, purification, structure elucidation and biological activity determination of new peanut phytoalexins. Unambiguous structural elucidation of new compounds was made possible by interpretation of their spectral properties. Lipophilicity of all compounds also was determined. The biological activity assay included the investigation of antifungal, antibacterial, anti-oxidative, anti-inflammatory and cytotoxic properties of new compounds. Additionally, the compounds were assayed for activity against mosquito larvae and as mammalian opioid receptor competitive antagonistic compounds. The relationship between chemical structure and biological activity of tested compounds was investigated. Other experiments were performed with abiotic elicitors to ensure that the newly discovered phytoalexins are not the products of fungal degradation. Such enzymatic activity could lead to a false positive detection of new compounds. 2010 is the second year for the collection of peanut leaf wax samples from the experimental control plots and peanut fields. Leaf wax was extracted with chloroform from pre-weighed leaves after determination of their surface area. Chloroform extracts were evaporated to dryness and kept frozen before analysis. Preliminary tests on chemical properties of waxes were performed.
Study of new defensive peanut stilbenoids and pterocarpenes produced by selected peanut genotypes. Detailed knowledge of peanut phytoalexin production may reveal mechanisms of peanut resistance to diseases; such mechanisms could be manipulated to improve natural field resistance of peanuts to pathogenic microorganisms. ARS researchers at Dawson, Georgia, isolated two new dimeric stilbenoids and two new pterocarpenes (extremely rare natural plant constituents) from fungal-challenged peanut seeds, and their structures were elucidated. The new pterocarpenes demonstrated strong antibacterial properties against gram-positive and gram-negative bacteria, particularly Bacillus subtilis and Staphylococcus aureus. Several new peanut stilbenoids showed anti-oxidative, anti-inflammatory and cytotoxic activity and were particularly inhibitory to the growth of the fungi Phomopsis obscurans, P. viticola and Botrytis cinerea, all considered major plant pathogens of economic importance worldwide. The discovery of peanut metabolites with a possible defensive role against pathogenic microorganisms may have an impact on peanut breeding programs designed to increase natural resistance to pests.
Evaluation of new strains of nontoxigenic fungi for biological control of aflatoxins in crops. Based on a laboratory peanut seed assay, ARS researchers at Dawson, Georgia, showed that four nontoxigenic strains of Aspergillus flavus exhibited a greater reduction of aflatoxins than the currently used strain that is incorporated into the biocontrol formulation afla-guard®. A patent disclosure has been submitted to assess the patentability of the potential biocontrol strains. These nontoxigenic strains singly or in combination may increase the effectiveness in controlling aflatoxin contamination using biological control.
Moore, G.G., Singh, R., Horn, B.W., Carbone, I. 2009. Recombination and lineage-specific gene loss in the aflatoxin gene cluster of Aspergillus flavus. Molecular Ecology 18(23):4870-4887.
Seifert, L.E., Davis, J.P., Dorner, J.W., Jaynes, W.F., Zartman, R.E., Sanders, T.H. 2010. Value Added Processing of Aflatoxin Contaminated Peanut Meal: Aflatoxin Sequestration During Protein Extraction. Journal of Agricultural and Food Chemistry. 58(9):5625-5632.
Sobolev, V.S., Neff, S.A., Gloer, J.B. 2009. New Dimeric Stilbenoids from Fungal-Challenged Peanut (Arachis hypogaea) Seeds. Journal of Agricultural Food & Chemistry. 58(2):875-881.
Horn, B.W., Moore, G.G., Carbone, I. 2009. Sexual reproduction in Aspergillus flavus. Mycologia. 101(3):423-429.
Dorner, J.W. 2010. Efficacy of a biopesticide for control of aflatoxins in corn. Journal of Food Protection. 73:495-499.
Condon, J., Sivakuman, G., Hubstenberger, J., Dolan, M.C., Sobolev, V.S., Medina-Bolivar, F. 2010. Induced Biosynthesis of resveratrol and the prenylated stilbenoids arachidan-1 and arachidan-3 in hairy root cultures of peanut: effects of culture medium and growth stage. Plant Physiology and Biochemistry. 48(5):310-318.