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United States Department of Agriculture

Agricultural Research Service

2009 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 naval 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, such as peanuts, corn, tree nuts, and in some countries, wheat. Aflatoxin contamination is a major food safety issue and is strictly regulated, resulting in it also being a significant international trade issue involving US agricultural products. The research in this project mainly involves development of environmentally friendly methods of preventing aflatoxin contamination. 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. 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 new method for collecting VOCs in situ and using electro-antennagram (EAG) and wind tunnel bioassays to determine which VOCs have bioactivity. This work is being done under a CRADA with a major tree nut stakeholder, in California. Efforts are also underway to use combinations of lure VOCs and pesticides in order to create an "attracticide." This strategy would function by using the volatiles to lure just the target insects to a specific trap containing a pesticide. This would serve as an alternative to a widely broadcasted pesticide spray.

The second approach by the scientists in the project 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.

1. Efforts to identify host-plant-volatiles (HPVs) affecting codling moth (CM) and navel orangeworm (NOW) behaviors. Navel orangeworm is the major insect pest of almond and pistachio, destroying millions of dollars of California tree nuts annually and California currently provides 100% of the nation's almonds alone and 80% of the world's supply of almonds. Currently, there are no truly effective lures to control NOW. New instrumentation was developed by ARS scientists in Albany, CA, for the comprehensive collection of volatile organic compounds (VOC) of tree nut orchards. Using this equipment a comprehensive list (nearly 100 compounds) of suspect attractant VOCs were collected and then tested on adult moths and in field trapping experiments. Discovery of any effective natural lures for NOW would have a major impact on California agriculture. By limiting the quantity of aflatoxin-producing fungi available in the ambient surroundings, the number of tree nuts infected with fungi would be decreased; thus, decreasing the overall levels of aflatoxin contamination.

2. Discovery of promising bacterial biological control agents for control of aflatoxin-producing fungi on corn. Aflatoxin is a carcinogenic fungi whose spores can be dispersed by wind. One way to control aflatoxin is to minimize wind dispersion of spores from infected soils. Wind tunnel experiments conducted by ARS scientists in Albany, CA, showed that application of two different Pseudomonas strains to soils infested with Aspergillus flavus spores significantly reduced the potential for windborne dispersal of fungal spores. Bacterial treatments resulted in the collection of 100- to 1000-fold fewer spores using a plate impact air sampler placed one meter away from the inoculated soil. These Pseudomonas strains were previously isolated from corn soils, and are currently being tested for biocontrol efficacy against A. flavus on corn, in collaboration with USDA-ARS scientists in Stoneville, MS. While laboratory assays show effectiveness, thus far, field bioassays are inconclusive.

3. Development of a culture medium for the mass production of a biocontrol agent of aflatoxin producing fungi. Maintaining the viability of the promising aflatoxin-producing fungi biocontrol yeast agent, Pichia anomala, in a formulated product is a great challenge. Biocontrol yeast cells are commonly dehydrated which can damage the cell membrane. A stable liquid formulation is highly desirable because the product can be directly diluted in water for spraying or dipping. Intra-cellular glycerol and arabitol are shown to increase under osmotic stress. ARS scientists in Albany, CA, developed a routine procedure for analysis of intracellular and extra-cellular sugar and polyol concentrations. The method has been used for developing stable liquid formulation of Pichia anomala WRL-076 and has vastly improved the output of agent for use in the field.

4. Attracticide developed against codling moth, a major pest of pome fruits and tree nuts. A novel attract-and-kill control tactic against codling moth larvae has been developed utilizing the pear ester kairomone attractant (PEK) in a micro-encapsulated formulation (MEC) as a spray adjuvant that augments-enhances the effectiveness of various insecticides when tank-mixed and sprayed together. The release rate of the evaporative PEK from applied microcapsules was experimentally measured by ARS scientists in Albany, CA, using the headspace capture techniques and chemical analysis. 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 insecticide sprays will enhance the effectiveness of insecticides and allow for lower, less costly, and more environmentally safe rates of insecticides to be confidently used for pest control.

6.Technology Transfer

Number of Active CRADAs1
Number of New Patent Applications Filed1

Review Publications
Beck, J.J., Merrill, G.B., Palumbo, J.D., O Keeffe, T.L. 2008. Strain of Fusarium Oxysporum Isolated From Almond Hulls Produces Styrene and 7-Methyl-1,3,5-Cyclooctatriene as the Principal Volatile Components. Journal of Agricultural and Food Chemistry. 56(23):11392-11398.

Beck, J.J., Merrill, G.B., Higbee, B.S., Light, D.M., Gee, W.S. 2009. In Situ Seasonal Study of the Volatile Production of Almonds (Prunus dulcis), var. 'Nonpareil' and Relationship to Navel Orangeworm. Journal of Agricultural and Food Chemistry. 57(9):3749-3753.

Palumbo, J.D., O Keeffe, T.L., Abbas, H.K. 2008. Microbial interactions with mycotoxigenic fungi and mycotoxins. Journal of Toxicology Toxins Reviews.27(3):261-285.

Last Modified: 10/6/2015
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