Inhibition of Tree Nut Contamination by Aflatoxins and Related Mycotoxins using Natural Products

Identify the natural constituents responsible for resistance of certain varieties of tree nuts to growth of aflatoxigenic strains of aspergillus. Isolate and identify novel metabolites in sclerotia of Aspergillus and develop analytical methods for such compounds in order to assess exposure levels of tree nut orchards to the fungus. Identify and develop methods to inhibit production of mycotoxins of phytopathogenic fungi endemic to orchard which debilitate tree nuts and exacerbate infection by Aspergillus. Identify biocompetitive micro organisms which supress growth of Aspergillus and aflatoxin formation.

Replaced 5325-42000-020-00D.

1. What major problem or issue is being resolved and how are you resolving it?

Contamination of tree nuts by aflatoxins produced on infection by the fungi Aspergillus flavus and Aspergillus parasiticus is a serious problem because of the stringent regulatory levels imposed for these toxins and the potential threat to human health. The current domestic guideline set by the FDA is 20 ppb total aflatoxins but the European Community has instituted a standard of 2 ppb aflatoxin B1 and 4 ppb total aflatoxins. Since the aggregate value of almonds, pistachios and walnuts crop is currently approaching $2 billion, and 60-70% of the crop is exported, this regulation constitutes a major threat to US exports of these crops. Research is being conducted to identify natural constituents of these nut crops that provide resistance to infection by Aspergillus and reduction in the ability of the fungus to produce aflatoxin, and also to identify yeasts that are biocompetitive towards Aspergillus.

2. How serious is the problem? Why does it matter?

Aflatoxin contamination of tree nuts is a serious problem because of the threat to human health and the regulatory constraints imposed on marketing crops that contain these toxins for human consumption. Of particular concern are the new EC regulations which will have a major impact on the US balance of trade because of the potential inability of many export shipments to conform to the aflatoxin levels imposed.

3. How does it relate to the National Program(s) and National Component(s)?

This research addresses the presence and potential for mycotoxins in crops which is not only a direct food safety problem but also threatens the competitiveness of US agriculture in the world market. The project is encompassed by National Program 108 (Food Safety), Program Component Mycotoxins (Fungal Toxins in Commodities).

4. What were the most significant accomplishments this past year?

A. Aflatoxin contamination in tree nuts is a serious problem. Laboratory experiments showed that the Tulare variety of walnut completely suppressed aflatoxin production and that the resistance factor is confined almost entirely to the thin, paper-like skin (pellicle) surrounding the kernel of the nut. This is the first example of a commercial variety of any crop plant known to be contaminated by aflatoxins that shows complete resistance. Since the pellicle is inherited from the maternal parent, it should be possible to develop new crosses with predictable, heritable natural resistance to aflatoxigenesis. B. Research was designed to identify optimal conditions under which biocompetitive yeasts will out-compete Aspergillus flavus. Yeasts were grown with a wide variety of carbohydrate and nitrogen sources and the growth of the yeast was quantified. It was found that the most effective yeasts can utilize a wide range of carbohydrates, which may improve their ability to compete with the fungi. This information provides basic information for cooperative field-tests of promising saprophytic yeasts as biocontrol agents against A. flavus in tree nut orchards. The DNA probe, pAF28, cloned from A. flavus genome has been useful to define the genotypes of A. flavus associated with corn. This probe was used to characterize isolates of A. flavus from almond, pistachio and walnut orchards. In this study, 48 isolates of A. flavus were analyzed and 25 fingerprint groups were distinguished. This information will be useful for prediction of A. flavus strains likely to cause aflatoxin contamination in tree nuts. A large-scale study of frequency of infection of tree nuts by Aspergillus and other fungi was initiated. Results showed that field collected nuts tended to be less infected than store bought nuts, and that nuts infected with A. flavus usually contained other fungi as well. This information will be used to predict the potential for aflatoxin contamination so that contaminated nuts can be removed from the process stream prior to mixing with uncontaminated product.

C. Significant Accomplishments/Activities that Support Special Target Populations.

None applicable

D. Progress Report.

Studies have shown that the stem-end of closed-shell pistachios provides a route for colonization by Aspergillus flavus which can result in exceptionally high aflatoxin levels in the nut meat. These results indicate that the practice of rehydration to facilitate cracking of closed-shell pistachios should be discontinued. The naphthoquinones present in the hulls of walnuts have been shown to significantly affect growth of A. flavus and production of aflatoxins, thus serving as a protection factor against contamination. Research has shown that the kernel of the Tulare walnut variety has natural resistance factors that suppress aflatoxin production. Saprophytic yeasts that are biocompetitive to Aspergillus flavus have been shown to utilize a wide range of carbohydrates as nutrient sources, suggesting that they will adapt well to field situations in tree nut orchards where they will be evaluated as biocontrol agents against A. flavus. A study of the frequency of infection of tree nuts by fungi has shown that nuts infected with A. flavus usually contained other fungi as well. This information will enable contaminated nuts in the process stream to be predicted more easily and facilitate their removal.

5. Describe the major accomplishments over the life of the project including their predicted or actual impact.

Kernels of the walnut variety, Tulare, have been shown to possess the ability to completely suppress aflatoxin formation in laboratory experiments. These results should permit the identification of specific resistance factors and establish the pattern of heritability since the parentage of this variety is known. Studies have shown that rehydration to facilitate cracking of closed-shell pistachios results in exceptionally high aflatoxin levels and that this process should be abandoned. A series of naphthoquinones which occur in walnuts have been shown to be inhibitors of aflatoxin production; this provides a useful tool to elucidate mechanisms of resistance. Saprophytic yeasts have been discovered that are biocompetitive to A. flavus. Successful field trials of these yeasts will provide a means to displace aflatoxin producing fungi from the tree nut environment.

6. What do you expect to accomplish, year by year, over the next 3 years?

Experiments will be conducted to identify the natural resistance factors to aflatoxin contamination present in Tulare variety walnuts and a pattern of heritability will be established. This information will be extended to almonds and pistachios. A comprehensive database of distribution of mycotoxin producing fungi in tree nuts will be developed. The genetic, physiological and morphological variation of A. flavus and A. ochraceous in tree nut orchards will be elucidated and their relationship to toxin production defined. The ecological relationship between biocompetitive yeast strains and Aspergillus flavus strains present in tree nut orchards will be studied to determine the factors essential for effective biocontrol. This project is being terminated. Its work will continue in the project 5325-42000-031-00D.

7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product?

Information has been developed which indicates that rehydration in order to facilitate cracking of closed-shell pistachios should not be employed because of the danger of aflatoxin contamination. The avoidance of this technique can be instituted immediately by processors.

8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)

32nd Annual Walnut Research Conference, January 26-28, 2000, Bodega Bay, CA. (Walnut growers, marketing board, and farm advisors).