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United States Department of Agriculture

Agricultural Research Service

2011 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 mentods 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. Replacing 5325-42000-031-00D (2/06).

3.Progress Report
This is the final report for the project 5325-42000-036-00D terminated January 2011. There was a period between the beginning of FY2011 and the termination date for the project in fall and winter. All planned experimentation for this project was completed. Some of this work served as a springboard for further research in a new project salient to the efforts of this project. The continuing efforts resulting from this project are outlined in the newly numbered project 5325-42000-038-00D.

PMR scientists developed methods for insect and microorganism control with regard to the aflatoxin contamination problem. MU scientists identified a number of host-plant volatiles (HPVs) that, as a blend, attract female and male navel orangeworm (NOW) at a higher rate than current commercial lures under field conditions. MU scientists also developed a new method for collecting HPVs. Much of this work was done under a CRADA with a major tree nut stakeholder in California.

MU scientists discovered a strain of yeast, Pichia anomala, that is a viable biocontrol microorganism against the fungi that make aflatoxin. This yeast shows a great deal of viability in tree nut orchards, has no human pathogenicity, is not phytotoxic, and can be sprayed directly onto the tree nut canopy. PMR scientists optimized the culture medium and are able to produce vast quantities of this organism.

Field trials to examine the efficacy of bacterial biocontrol strains for controlling aflatoxin and fumonisin on corn showed that bacterial treatments had no effect on mycotoxin levels in treated corn.

1. Using bacteria to control fungal infection in crops. Food safety will improve by developing methods to reduce mycotoxin contamination of food commodities. Research focused on developing methods using biological control to control mycotoxin-producing fungi. ARS scientists at Albany, CA, demonstrated reduction of fungal populations in soil co-inoculated with biocontrol bacteria, leading to reduced spore production and potential for reduced infection of corn via windborne spores. The use of such bacteria could lower the need for fungicides, which may be harmful to the environment.

2. Safe, natural yeast reduces aflatoxin contamination. Aflatoxin is a natural carcinogen that can contaminate tree nuts. Reducing this contamination is necessary to reduce economical losses due to tight controls on exports to other countries. ARS scientists in Albany, CA, in collaboration with CRADA partners, discovered one yeast species, Pichia anomala that reduces infection of tree nuts by aflatoxin-producing fungi. They have developed production methods for this yeast, as well. In addition, they are using molecular techniques to identify other species of yeast that are natural residents of tree nut orchards. These other yeasts may prove to be good biocontrol agents, as well. Effective biocontrol will be a safe way to enhance food safety.

3. Control of pest insects of tree nuts using natural lures. Insect damage of tree nuts allow contamination by aflatoxin. ARS scientists in Albany, CA, in collaboration with CRADA partners, have developed methods for collecting, identifying and bioassaying natural volatiles that influence behavior of insect pests of tree nuts. They found blends of natural volatiles that can be used to lure these pests to traps; two in particular include codling moth (CM) and navel orangeworm (NOW). One lure, the pear ester, enhances pheromone mating disruption of CM and pear ester microencapsulated (PE-MEC) formulation disrupts normal behavior of larval CM. Several blends of volatiles show promise for control of NOW. Some of these blends show better activity in the field, as lures, than currently available commercial products. Use of natural lures to control tree nut insect pests can reduce the risk of loss due to insect damage and concomitant contamination of aflatoxin.

Review Publications
Roitman, J.N., Merrill, G.B., Beck, J.J. 2011. Survey of ex situ fruit and leaf volatiles from several Pistacia cultivars grown in California. Journal of the Science of Food and Agriculture. 91(5):934-942.

Palumbo, J.D., O Keeffe, T.L., Vasquez, S.J., Mahoney, N.E. 2011. Isolation and identification of ochratoxin A-producing Aspergillus section Nigri strains from California raisins. Letters in Applied Microbiology. 52:330-336.

Palumbo, J.D., O Keeffe, T.L., Mcgarvey, J.A. 2011. Incidence of fumonisin B2 production within Aspergillus section Nigri populations isolated from California raisins. Journal of Food Protection. 74:672-675.

Hua, S.T., Brandl, M., Hernlem, B.J., Eng, J.G., Sarreal, S.L. 2010. Fluorescent viability stains to probe the metabolic status of aflatoxigenic fungus in dual culture of Aspergillus flavus and Pichia anomala. Mycopathologia. Available:

Light, D.M., Beck, J.J. 2010. Characterization of microencapsulated pear ester, (2E,4Z)-ethyl-2,4-decadienoate: a kairomonal spray-adjuvant against neonate codling moth larvae. Journal of Agricultural and Food Chemistry. 58(13):7838-7845.

Last Modified: 5/27/2015
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