2011 Annual Report
1a.Objectives (from AD-416)
Enhance value and utilization of low value/underutilized crops and crop co-products through discovery and purification of novel, constituent antifungal compounds and determine commercial potential of discovered antifungals.
1b.Approach (from AD-416)
Barley and cotton 3-day old cotyledons will be extracted aqueously, as will okra fruit, and peanut and rice hulls. Filtered, freeze-dried extracts will be tested for fungicidal properties. Active compounds will be purified by HPLC/MS and activity monitored with bioassays using Aspergillus flavus and Fusarium oxysporum. NMR will determine chemical structure of antifungal compounds. Novel compounds will be patented and all active compounds will be tested for their antifungal spectra of activity. Commercial potential of compounds as agricultural fungicides and medical antifungals will be determined by collaborators. Discovered protein antifungals will be cloned. Construct genes will be incorporated into gene expression systems to obtain a protective effect against fungal pathogens.
Progress was made on all research areas. Research identified unique volatile compounds produced by aflatoxin-producing Aspergillus (A.) flavus grown on corn. Data was used by our stakeholder, to develop a sensor for aflatoxin-producing A. flavus growth in corn storage facilities. A new volatile trapping system and updated compound library funded by a grant were installed in our instrument [gas chromatograph/mass spectrometer (GC/MS)] to identify toxigenic and non-toxigenic A. flavus isolate volatiles when grown on wet (20% moisture) non-sterile corn. Volatiles varied between all isolates with some being unique for toxigenic isolates. Aflatoxin levels were compared to the presence of unique volatiles to determine if they could act as aflatoxin indicators. In another project, trans-2-hexenal, a commercially available soybean volatile, pumped intermittently for a week inhibited A. flavus growth and aflatoxin production on sterile, wet corn in a bench model system. Due to the unexpected extensive use of the GC/MS on A. flavus volatile identification, the next work stage began in mid FY 2011. Our goal is to develop safe methods to protect stored corn from fungal growth and toxin contamination. In vitro, commercial volatile plant compounds, a-pinene and limonene, have good antimicrobial activity. Results showed excellent antifungal activities of mixed wheat histones H1-H4 and purified H1. Future work will increase microbe type and plant compounds studied. Our goal is to use active, safe, plant compounds in coatings to protect seeds and/or citrus from fungal rot. Progress was made in our study of visible blue (not UV) light antimicrobial properties. Studies showed commercial light emitting diodes (LEDs) significantly reduced bacterial viability. Some tested bacteria possess repair systems. Tests showed incubation temperature after LED exposure played a significant role in viability loss to some bacteria, indicating temperature affects the repair system. LEDs from two manufacturers were used. One array, producing blue light mixed with other colors, was more active than an array producing “pure” blue light and suggests minor levels of “contaminating” non-blue light increased lethality to certain bacteria. Blue light alone does not significantly reduce fungal viability. However, significant fungal viability loss occurred when incubated with the photosensitizer, FD&C red #3 food dye, and exposed to blue light. Studies were begun on the antimicrobial properties of a red light array (LEDs, 625nm). Red light alone was inactive so synergistic studies were begun with safe antimicrobials or combined red and blue light treatments. Safe, visible light may provide a novel method to sanitize surface areas, processed crops, or baked goods from microbial growth.
Identification of secondary metabolic volatile compounds unique for toxigenic Aspergillus (A.) flavus grown on corn. Aspergillus flavus, a naturally-occurring fungi on corn can grow and produce aflatoxin, which causes liver cancer, when the corn becomes wet. Annually, millions of dollars are lost to the United States corn and oilseed industries due to aflatoxin contamination. Current detection of this toxin requires labor intensive, time-consuming testing of corn in a laboratory, which reduces the ability of grain handlers to remove the contaminated corn and prevent the contamination from spreading to wholesome corn. Agricultural Research Service researchers (Food and Safety Research Unit, Southern Regional Research Center, New Orleans, LA) provided data used by a stakeholder to build an electronic sensor, still under development, to warn grain handlers of the growth of this fungus and aflatoxin contamination of stored corn. The goal of this cooperative effort is the manufacturing and sale of a “real-time” sensor and sale to grain handling facilities to warn them of Aspergillus flavus growth and aflatoxin production in stored corn.
De Lucca II, A.J., Boue, S.M., Carter-Wientjes, C.H., Bland, J.M., Bhatnagar, D., Cleveland, T.E. 2010. Volatile profiles of toxigenic and non-toxigenic Aspergillus flavus using SPME for solid phase extraction. Annals of Agriculture and Environmental Medicine. 17:301-308.
De Lucca II, A.J., Pauli, A., Schilcher, H., Sien, T., Bhatnagar, D., Walsh, T.J. 2011. Fungicidal and bactericidal properties of bisabolol and dragosantol. Journal of Essential Oil Research. 23:47-54.