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United States Department of Agriculture

Agricultural Research Service

2008 Annual Report

1a.Objectives (from AD-416)
The overall objective of this project is to apply the Pichia anomala yeast product to pistachio orchards early in the season prior to June 15 to be followed by 'atox' technology. Measure the reduction of recoverable Aspergillus flavus spores in treated plots or use some other appropriate measure of reduced colonization of natural substrates. Will develop commercially viable methods for control of fungal and insect pests which contribute to pre-harvest aflatoxin contamination of tree nuts. The methods are to be environmentally benign and not harmful to humans. To control fungi, there are two main objectives. The first objective for this project is to control mycotoxin-producing fungi using bacteria. The second biocontrol effort is well underway and involves the biological control of A. flavus in tree nut orchards using the saprophytic yeast Pichia anomala. The yeast will also be examined to determine its effectiveness against other fungal pathogens, e.g., Fusarium spp., Penicillium spp. The third objective of this project is also an ongoing research effort. This objective entails development of semiochemical-based low-risk control strategies against key insect pests of tree nuts. Insect feeding damage is associated with the invasion of microbial pathogens and mycotoxin contamination.

1b.Approach (from AD-416)
Develop methods to control insect pests and toxic fungi of tree nuts. Insects include navel orangeworm, codling moth and peach twig borer. Feeding damage by these insects leads to infection by aflatoxigenic aspergilli. Control methods for insects are to be environmentally benign and employ semiochemicals to disrupt insect behavior. Control of toxic fungi focuses on biological control using competitive or antagonistic microorganisms. These microorganisms include either yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post harvest environments.

3.Progress Report
Aflatoxin is a highly carcinogenic compound produced by certain fungi that can infect a wide variety of edible agricultural products. Aflatoxin contamination is a major food safety issue and is strictly regulated, resulting in it also being a significant international trade issue. USDA scientists in the Plant Mycotoxin Research Unit, Albany, CA, are tackling this problem by developing methods that involve insect and microorganism control, namely methods that fall under the general umbrella of what is termed "biological control." MU scientists clearly showed that insect feeding damage can lead to aflatoxin contamination. This was shown in experiments involving the navel orangeworm (NOW), the major insect pest of almond. NOW was able to carry spores of aflatoxin producing fungi on hairs that cover its body. Moreover, when NOW created feeding wounds on almond kernels, these spores were transferred to these wounds, increasing the potential for aflatoxin contamination. MU scientists are attempting to control pest insects by identifying host-plant volatiles (HPVs), also termed volatile organic compounds (VOCs). These HPVs could be used as lures to traps or as a means of confusing or distracting the insects from locating the host-plant. MU scientists have made substantial progress in this area by developing a significant HPV lure to the codling moth, a major pest of walnuts and pome fruits. MU scientists are also attempting to identify HPVs that could be used against the navel orangeworm (NOW), one of the most destructive insect pests to the tree nut industry. Development of a lure to NOW has been elusive, to date. In this regard, MU scientists have developed new methods of trapping test volatiles in the field and are using sophisticated electro-physiological tools to hone-in on the HPVs of most significance. This work is being done under a CRADA with a major tree nut player, Paramount Farms, in California. The second approach by the scientists in the CRIS for controlling aflatoxin contamination is use of competitive micro-organisms against the fungus that makes aflatoxin. In this regard, there are two approaches. MU scientists have discovered a potentially promising strain of yeast, Pichia anomala. This yeast shows a great deal of viability in tree nut orchards, has no human pathogenicity (unlike current atoxigenic strains of the fungus that produces aflatoxin that are currently being promoted as the best biological control agents), is not phytotoxic, and thus can be sprayed directly onto the tree nut canopy. The research is being done in collaboration with scientists in the Dept. of Pomology, Univ. of California, Davis. A second biocontrol approach involves identification of natural soil bacteria in tree nut orchards that could show promise as biological control agents against fungi in soil. In this project, a number of bacteria have been identified and are currently being tested in corn fields by collaborators in the Crop Genetics and Production Research Unit Stoneville, Mississippi. This work supports NP 108 Component 2.1.5.

1. Navel Orangeworm Activity Can Promote Aflatoxin Contamination of Almonds. A major insect pest of almonds, the navel orangeworm, has been found by Plant Mycotoxins Research scientists to be a major contributor to promoting infection of almonds by the fungus that produces aflatoxin. This insect can carry the spores of this fungus (Aspergillus) to the almond kernel. The spores can then infect the almond through feeding wounds produced by the insect. Funding for this pilot study was provided by the Almond Board of California (ABC). This accomplishment relates to NP108, Component 2, Problem Statement B, Crop/Fungal/Insect/Toxin Relationships.

2. Bacteria as Biological Control agents of aflatoxin producing fungi. Aflatoxin contamination of agricultural crops, such as corn, tree nuts, peanuts and cotton seed, can result in serious economic losses on an annual basis. One approach that shows some promise of success in controlling contamination is use of other microbes as biological control agents against aflatoxin producing fungi. Strains of two bacteria were discovered by ARS scientists in the Plant Mycotoxins Research Unit in Albany, CA that reduced aflatoxin producing fungi by 10 to 100 fold in soil from corn fields suggesting that environmental and nutritional conditions in this soil were not conducive to aflatoxin production. This work was partially done in collaboration with USDA-ARS in Stoneville, MS for field trials on corn. This accomplishment relates to NP108, Component 2, Problem Statement E, Biocontrol Technologies.

3. Yeast as Biological Control Agent of Aflatoxin Producing Fungi. Aflatoxin contamination of agricultural crops, such as corn, tree nuts, peanuts and cotton seed, can result in serious economic losses on an annual basis. One approach that shows some promise of success in controlling contamination is use of other microbes as biological control agents against aflatoxin producing fungi. Application of a yeast by ARS scientists in the Plant Mycotoxins Research Unit in Albany, CA resulted in significant reductions in cluster loss and significant increases in harvestable yield of pistachios. Yield for the yeast treatment was 12% greater than for the control. The yeast also effectively controlled a waterborne bacterial contaminant, and may be useful as a control measure against human pathogenic bacterial contaminants in the field. This research shows that this yeast may be a promising biological approach to controlling aflatoxin producing fungi directly on pistachios, without harming the quality of the harvested nuts. This accomplishment relates to NP108 Component 2, Problem Statement E, Biocontrol Technologies.

4. Use of Natural Chemical Lures and Viruses to Control Insect Pests of Tree Nuts. Navel orangeworm is the number one insect pest of almonds and pistachios. A new large-scale, ambient and in-situ volatile organic chemical (VOC) collection system. Allows for monitoring/collection of ambient orchard VOCs, in-situ single cultivar VOCs of leaves and/or almonds, and in-situ damaged almond VOCs. ARS scientists in the Plant Mycotoxins Research Unit in Albany, CA implemented, in cooperation with Paramount Farming Company (PFC), both ambient and damaged VOC collections of almonds. Several NOW attractant VOCs were identified and a novel attract and kill (AK) – attracticidal control tactic against codling moth (CM) larvae - method has been developed utilizing the pear ester kairomone attractant (PEK) in a micro-encapsulated formulation (MEC) as a spray adjuvant that augments effectiveness of various insecticides when tank-mixed and sprayed together. The benefit is to achieve the longest time-exposure by crawling larvae to leaf-surface applied insecticides. Data suggests that inclusion of the PEK-MEC adjuvant in the insecticide sprays will enhance the effectiveness of insecticides and allow for lower, less costly, and more environmentally safe rates of insecticides for CM control. This accomplishment relates to NP108 Component 2, Problem Statement B, Crop/Fungal/Insect/Toxin Relationships.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of Active CRADAs1
Number of Non-Peer Reviewed Presentations and Proceedings4

Review Publications
Palumbo, J.D., O Keeffe, T.L., Abbas, H.K. 2007. Isolation of maize soil and rhizosphere bacteria with antagonistic activity against Aspergillus flavus and Fusarium verticillioides. Journal of Food Protection. 70(7):1615-1621.

Beck, J.J., Higbee,B.S., Merrill, G.B., Roitman, J.N. 2008. Comparison of volatile emissions from undamaged and mechanically damaged almonds. Journal of the Science of Food and Agriculture.88(8):1363-1368

Beck, J.J., Smith, L., Merrill, G.B. 2008. In Situ Volatile Collection, Analysis, and Comparison of Three Centaurea Species and Their Relation to Biocontrol with Herbivorous Insects. Journal of Agriculture and Food Chemistry.56(8):2759-2764.

Wilson, K.A., Beck, J.J. 2007. Complete Proton and Carbon Assignment of Triclosan via One- and Two- Dimensional Nuclear Magnetic Resonance Analysis. The Chemical Educator.12(5):338-342

Chou, S., Everngam, M.C., Beck, J.J. 2008. Allelochemical phenolic acids from Gypsophila paniculata. Journal of Undergraduate Chemistry Research.7(2):40-42.

Last Modified: 10/13/2015
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