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United States Department of Agriculture

Agricultural Research Service

2011 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
Refinement of the biological control formulation Afla-Guard®, which consists of barley coated with oil containing spores of a non-toxigenic strain of Aspergillus flavus, was completed. Oils representing a range of oxidative stabilities were tested for their effects on the shelf life of Afla-Guard®. Of the oils tested, mineral oil was optimal and extended the shelf life to over 19 months. Afla-Guard® was also tested on peanuts in Nicaragua and the 98% reduction in aflatoxin contamination provided the first strong evidence that Afla-Guard® can be used effectively in other parts of the world. Under an experimental use permit by the Environmental Protection Agency (EPA), Afla-Guard® was tested for its effectiveness on corn in southern Texas. Aflatoxin contamination of corn was reduced by 85-88% over a two-year period, and EPA subsequently approved its use on corn. The biocontrol technology is now labeled for commercial use on both peanuts and corn.

A peanut laboratory assay was developed in which viable fungus-free peanut seeds are wounded and inoculated with soil containing natural fungal populations. This assay showed that peanut seeds are invaded at extremely low soil densities of A. flavus and that infection is optimal at specific combinations of temperature and seed water activity. The assay also was used to evaluate eight non-toxigenic A. flavus strains for biological control of aflatoxins in peanuts. Four of those strains were superior to NRRL 21882, the non-toxigenic strain in Afla-Guard®, in reducing aflatoxin contamination. During the course of the project, the sexual stages of the three major aflatoxin-producing fungi (A. flavus, A. parasiticus and A. nomius) were discovered. Approximately 3500 progeny strains from laboratory crosses were generated for genetic analysis of recombination overall and with respect to the aflatoxin gene cluster. Several new efficient analytical methods for mycotoxin and peanut phytoalexin analyses were developed. The methods allow for significant reduction in the cost of analyses for aflatoxins and for quantitative determination of peanut stilbenoids. The latter method helped elucidate the complex dynamics of phytoalexin synthesis in peanut seeds under invasion by A. flavus. Nine novel stilbenoid phytoalexins and two new pterocarpenes were discovered in fungal-challenged peanut seeds, and seven new stilbenoids were described from root mucilage. Field experiments demonstrated a direct association between peanut resistance to major fungal and viral diseases and levels of phytoalexin production by seeds. The study of biological activity of peanut phytoalexins revealed strong antifungal (in stilbenoids) and antibacterial (in pterocarpenes) activities, which resulted in the elucidation of their structure-bioactivity relationship and prediction of their behavior in the host-pathogen interactions. This research also showed strong antioxidant, anticancer, and anti-inflammatory properties of several stilbenoids in a panel of human cell lines. This CRIS project expired 12-14-2010. It was relaced by a bridge #6604-42000-009-00D while this project is in review by ONP.

Last Modified: 8/1/2015
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