1a. Objectives (from AD-416)
The overall objective of this project is to develop commercially viable methods for the control of pathogens in tree nuts (almonds, pistachios and walnuts) and raisins which lead to foodborne illness in humans and animals. This includes both the control of fungal pathogens (mycotoxins) as well as bacterial pathogens such as E. coli and Salmonella. One approach will be the development of biocontrol agents using bacteria, yeast and non-toxigenic Aspergillus carbonarius. In addition, a better understanding of organic and conventional farming systems will provide new insights on mycotoxin control. The specific objectives for the period covered by this project plan are as follows: Objective 1: Define the critical control points for pathogen contamination during the production stream. Place particular emphasis on agricultural water sources including dairy waste water. Using both cultural and non-cultural based methods we will identify the points in the developmental process, as well as the processing cycle where tree nuts are most likely to become contaminated with human pathogenic bacteria and mycotoxigenic fungi. Objective 2: Evaluate the microbial ecology of organic v. conventional practices. Although several surveys have reported that consumers equate organically grown food stuffs with higher levels of food safety, little is known about how these practices affect the microbial population structure or mycotoxin levels in tree nuts. We will address the influence of phyllosphere microbial community on the population diversity of A. flavus in tree nut orchards, and A. carbonarius on grape surfaces in both farming systems. Objective 3: Delineate the factors affecting cross-contamination during processing and develop a potential intervention strategy during storage. A number of experiments point to a strong possibility that cross-contamination of toxigenic fungi is possible during processing and storage, although no research appears to have been done on the transfer in actual (not laboratory) processing and storage conditions. Development of novel approaches to prevent the growth of storage fungi and production of harmful toxins is a high priority in the almond industry. Edible films and coatings (EFC) containing antimicrobial natural compounds will be tested for their efficiency to reduce mycotoxin in stored almonds. Objective 4: Develop biological-control/intervention technologies using competitive or antagonistic microorganisms such as yeasts or bacteria that can be mass-produced and effectively utilized in a variety of pre- or post-harvest environments. Bacterial and yeast biocontrol agents will be tested in almond orchards and vineyards to control A. flavus and A. carbonarius, as well as human pathogenic bacteria, E. coli and Salmonella. EPA registration of the patented yeast, Pichia anomala will be pursued for commercial application. Methods to enhance the biocontrol efficacy will be developed. The flowchart shown in Figure 1 illustrates the integration of the four objectives to achieve the ultimate goal of eliminating mycotoxins and human bacterial pathogen contamination (i.e. Salmonella, E. coli O157:H7, etc.) in tree nuts and raisins.
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. Replacing 5325-42000-036-00D (1/11).
3. Progress Report
This report documents progress for the parent project 5325-42000-038-00D ENVIRONMENTAL AND ECOLOGICAL APPROACHES TO ELIMINATE FUNGAL CONTAMINATION AND MYCOTOXIN PRODUCTION IN PLANT PRODUCTS which started Feb 2011 and continues research from project 5325-42000-036-00D BIOLOGICAL CONTROL OF INSECTS AND MICROORGANISMS TO PREVENT MYCOTOXIN CONTAMINATION. In cooperation with scientists from University of California, three organic and three conventional almond orchards were identified and selected as sources for almonds for the research. Immature almonds from all 6 sites have already been collected and additional samples will be collected as the nuts mature on the trees. Fungal populations from raisins produced under different management practices have been isolated. Isolated strains have been identified to the species level, and the ability of each strain to produce mycotoxins (ochratoxin and/or fumonisin) are ongoing. Small-scale field trials to determine efficacy of bacterial biocontrol agents on corn for reducing aflatoxin contamination showed that the bacterial strains that performed best in laboratory assays did not produce measurable reduction of aflatoxin on corn. Collection and identification of black Aspergillus isolates from vineyard samples of raisins. These isolates are currently being screened for ochratoxin and fumonisin production. Frequency of species and mycotoxin production within each sample will be analyzed relative to production method to determine whether patterns of correlation exist. Almond nut samples have been obtained from commercial sources and half of them have been “spiked” artificially with aflatoxin. Mixing experiments are ready to proceed to determine the mode of aflatoxin cross contamination. A variety of films have been fabricated and screened in order to find films that are effective in preventing fungal growth. Edible tomato and apple based films containing cinnamon oil, allspice oil, oregano oil, garlic oil, bay laurel oil, bay leaf oil, cedarwood oil, lemomgrass oil, and thyme oil are being tested. Interactions of yeast cells with Aspergillus flavus or cell walls of the fungus were investigated. The study is to identify factors which may be used to enhance the efficacy of biocontrol activities of the yeast, Pichia anomala. Yeast cells grown in the presence of fungi or fungal cell walls, were collected for gene expression studies. Parameters influencing real time polymerase chain reaction (RT-PCR) were examined. A procedure to determine intracellular sugars and polyols by high pressure liquid chromatography (HPLC) analysis is being developed.
1. Evaluation of antifungal films for use in almond storage. Usually fungal growth on stored almonds in the bin starts near the lid due to moisture condensation. The use of films as carriers of natural antimicrobials (such as plant essential oils) constitutes an alternative approach for external protection of almonds, especially raw almonds, to reduce surface microbial populations. ARS scientists at Albany, CA, have developed a method to screen edible films which can inhibit both aflatoxin production and fungal growth. Several edible films have been identified so far. These films can be easily applied on the lid of the container and protect raw almonds during transport.
2. Molecular mechanisms of biocontrol yeast, Pichia (P.) anomala. Understanding the modes of action is important for maximizing the potential use of microbial biocontrol agents. ARS scientists at Albany, California, have identified two genes in P. anomala that produce enzymes that break down the cell wall of Aspergilles (A.) flavus. They discovered that expression of these genes can be increased four to six-fold upon exposure to A. flavus. This finding validates P. anomala as a promising biocontrol agent against A. flavus.
3. Improve shelf-life of biocontrol yeast, Pichia anomala. Maintaining the viability of this biocontrol agent in formulated products is a challenge. A wet formulation has been developed by ARS scientists in Albany, CA, by adding certain compounds to the formulation. The shelf-life of this formulation was up to fourteen months with very little change in viability of yeast cells stored at 40c. The results were validated by flow cytometry analysis and florescent microscopy using viability stains. A stable liquid formulation is highly desirable because most of the bicontrol products are dispersed in water and delivered by spraying or dipping to protect agricultural and horticultural crops.