1a. Objectives (from AD-416):
The overarching goal of this project is to develop methods to improve the safety of our food with regard to contamination by microbial toxins, mainly mycotoxins. In this regard, the project has a series of interconnected objectives. These objectives and their interrelationships are described as follows: Objective 1: Develop detection methods for Volatile Organic Compounds (VOCs) to pinpoint food contaminants. Develop methods for identifying specific, "signature," volatile organic compounds (VOCs) as telltale indicators of microbial, mycotoxin contamination or insect infestation. This VOC detection will be applied to Objective 2. Objective 2: Develop methods for real-time detection of pathogen or insect contamination and toxins. Telltale VOCs will be used as real-time "signals" for detecting the presence of pathogens, mycotoxin-producing fungi or insects in crops or crop products, in post harvest storage or processing environments. This detectability will permit optimization of any intervention measures that are needed; which will be developed in Objective 3. Objective 3: Develop intervention technologies using plant VOCs / natural products as tools for the control of pathogens and insects. Specifically target the genetic control mechanisms through the use of chemosensitization and formulation development. Objective 4: Investigate the biochemical and genetic regulation of toxin production within fungal communities, with special emphasis on communication. Investigate the factors controlling aflatoxin catabolism and identification of the catabolic products.
1b. Approach (from AD-416):
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 genes involved in triggering mycotoxin biosynthesis using high-through put bioassays. Assays involve use of deletion mutants, gene knockouts and complementation analysis. Discover natural compounds that disrupt functionality of gene targets identified. Develop biosensors for detecting toxic fungi in pre- and post harvest environments.
3. Progress Report:
Considerable progress has been made towards achieving each objective during the present reporting period and much of the work has been published, in print, or under preparation. Some of the major progress includes: extensive field trapping studies in commercial almond orchards with a blend of host plant volatiles from damaged and fungal-contaminated almonds which has consistently demonstrated significantly higher capture rates during monitoring studies of the insect pest navel orangeworm than the current commercial standard; volatile profiles of fungal contaminated almonds and pistachios have been performed and are being investigated as signature volatiles for the detection of aflatoxin-contaminated tree nuts; a highly portable gas chromatograph-mass spectrometer (GC-MS) has been acquired through an agreement with a manufacturer and is being applied toward project goals; a yeast-generated volatile has been discovered that inhibits the production of aflatoxin by Aspergillus flavus; and, the natural product kojic acid was found to mediate chemosensitization of certain Aspergillus species.
1. Fungal spores on tree nut products as early warning volatile signaling. The detection and removal of aspergilli-contaminated almonds and pistachios is a high priority for the California tree nut industry. ARS Researchers at Albany, California, discovered that fungal spores common to tree nut orchards emit distinctive volatiles during the transition from resting to developing. This transition indicates an active period for fungi in which the production of aflatoxin may soon occur. These volatiles provide early-warning of contamination and thus a means for intervention prior to aflatoxin production. This discovery will allow for the immediate in-field removal of the contaminated product decreases risk of the present fungal growth as well as subsequent contamination of other materials. Current aflatoxin monitoring requires calcium treatment a week before a batch can be declared safe or contaminated.
2. Natural compounds for use as antifungal chemosensitizers. ARS Researchers at Albany, California, discovered that a set of natural secondary metabolites, such as product(s) of some filamentous fungi infecting tree nuts or of plants, greatly increase the effectiveness of conventional antifungal agents against fungal pathogens. Co-application of natural chemosensitizers with hydrogen peroxide, polyene or strobilurin fungicides significantly enhances the effectiveness of these antifungal agents against mycotoxigenic phytopathogens or human pathogens (yeast, filamentous). With the current increase of fungal resistance to fungicides and drugs, the ability to enhance a drug's efficacy while at the same time lowering the dose decreases the chance of a fungus developing resistance. Results thus far show that chemosensitization could also be used for identifying vulnerable molecular target(s) in different fungi, a crucial factor for developing strategies towards antifungal drug formulation.
3. A portable gas chromatograph-mass spectrometer (GC-MS) for the rapid, non-invasive, and early detection of aspergilli on almonds and pistachios. Current methods for the detection and removal of fungal-contaminated tree nuts require the removal and destruction of large amounts of product, and may take up to a week to obtain results regarding actual contamination levels. ARS Researchers at Albany, California, have recently formed an agreement with a private company that manufactures portable GC-MS instruments. Through a jointly written grant, the ARS Researchers have acquired a portable GC-MS for the detection of early warning signaling volatiles. Congruent with the accomplishment of discovering the early-warning volatiles emitted from spores, the portable GC-MS provides researchers the means to perform, in near real-time, the collection, analysis, and identification of volatiles emitted by contaminated almonds and pistachios. These "hot spots" of fungal contamination can be immediately removed from the stockpile or transit container.
Kim, J.H., Chang, P., Chan, K.L., Faria, N.G., Mahoney, N.E., Kim, Y., Martins, M.L., Campbell, B.C. 2012. Enhancement of activity of commercial antifungal agents by kojic acid. International Journal of Molecular Sciences. 13:13867-13880. DOI:10.3390/ijms131113867.