Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. Accomplishments 01 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 preven the contamination from spreading to wholesome corn. Agricultural Resear 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 a sale of a �real-time� sensor and sale to grain handling facilities to wa them of Aspergillus flavus growth and aflatoxin production in stored cor
Impacts
(N/A)
Publications
- 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.
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Progress 10/01/09 to 09/30/11
Outputs
Progress Report 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. 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. This is the final report for the bridging project 6345-41000-101-00D terminated September 30, 2011, when the new in-house project 6435-41000- 107-00D commenced. Progress was made on all planned research. Plant volatile compounds were tested and found lethal to Aspergillus (A.), Fusarium (F.) and Penicillium (P.) species that are problems in post- harvest corn (A. flavus, F. verticillioides) and citrus (P. digitatum, P. italicum). Initial studies in a small model of stored corn showed that volatilized compound trans-2-hexenal (produced by soybeans when infected with A. flavus) intermittently pumped into sterile corn inoculated with A. flavus prevents the growth of this fungus and subsequent aflatoxin production. New project research will build upon the study of this and other safe volatiles to prevent fungal, especially toxigenic fungi that are problems on corn, and bacterial growth on wet, non-sterile corn in our stored model system. Work was nearly completed on the identification of unique secondary metabolic volatiles produced by toxigenic strains of A. flavus, as compared to non-toxigenic strains and corn controls, when grown separately on sterile and non-sterile corn. Our stakeholder, Sensor Development Corporation, used this data to develop a real-time sensor which is undergoing Beta (secondary) testing by a large grain company in a silo. The work will continue in the new project with studies of unique volatile compounds produced by toxigenic strains of Fusarium verticillioides when grown on corn. Basic research into the antifungal and antibacterial properties of selected, safe, inexpensive plant compounds having volatile properties, continued to identify promising compounds to be used in the new project for testing in (1) the small scale corn silo model; (2) prevention of the bacteria Leuconostoc mesenteroides in raw sugar cane juice; and (3) safe, food grade edible films. Initial in vitro studies of the antimicrobial properties of blue light (470 nm peak) were completed and found to significantly reduce bacterial and fungal viability. Accomplishments 01 Detection of unique volatiles produced by toxigenic Aspergillus (A.) flavus could be used to protect stored corn from fungal growth. Aspergillus flavus, commonly found on corn, produce aflatoxins (the most potent natural hepatocarcinogens known) and contaminated commodities are banned for sale at aflatoxin concentrations of 20 ppb or higher. When conditions are conducive, the fungus readily grows on stored corn and renders it unsafe. ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, identified unique volatiles produced by toxigenic A. flavus isolates. This led to t development of a sensor by our Stakeholder, Sensor Development Corporati Detection of these volatiles could be used by companies to remove the contaminated corn batch before it contaminates wholesome corn, thus increasing the supply of wholesome corn for national and international u 02 Use of safe, commercially available plant compounds to protect post- harvest food and feed begins with basic in vitro research into their antifungal and antibacterial properties. Some safe, inexpensive, plant produced antimicrobial compounds including some with volatile properties which could be employed in the volatile state to protect stored post harvest commodities (e.g., corn) from infection while non-volatile antimicrobials could be incorporated into food grade films. ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, are investigating their antimicrobial properties (1) against toxigenic fungi (Aspergillus (A.) flavus, Fusariu (F.) verticillioides, F. graminearum) found on grains, as well as fungi (Penicillium (P.) digitatum, P. italicum) that cause rot in post-harvest citrus. Such commercially available compounds displaying significant antifungal and antibacterial (e.g., against the bacteria Escherichia col Bacillus (B.) subtilis, B. atrophaeus) activity below 20�M include compounds, namely bisabolol, dragosantol, wheat histones, carvacrol, citral, and linalool. Results of this work will be used to develop (1) volatile treatment to prevent the growth of all fungi, especially mycotoxigenic fungi, and spoilage bacteria on wet non-sterile corn in ou storage model and (2) antimicrobial food grade films. 03 Visible blue light (470 nm) is antimicrobial with potential use in food protection. Ultraviolet (UV) light (10-400 nm) has antimicrobial properties but is a Group 1 carcinogen which causes cancer in humans. However, visible blue light (450-475 nm) is not carcinogenic and has antibacterial properties. ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, conducted experiments showing light-emitting diodes (LED) (470 nm) produ blue light that is lethal for bacteria that are problems in sugar cane factories and aquaculture. In vitro, blue light at an exposure of 150 J/cm2 is significantly lethal to the bacteria Leuconostoc mesenteroides which converts sucrose (type of table sugar) in cane juice to dextrans (complex molecule made up of glucose). It is also significantly lethal (at = 10J/cm2)to bacteria Aeromonas hydrophila, a pathogen of catfish in aquaculture ponds as well as Bacillus (B.) atrophaeus, a surrogate used study antimicrobials targeting B. anthracis, the microbe causing anthrax Alone, blue light was not found to be fungicidal, though fungal viabilit was reduced in the presence of photosensitizing compounds such as the fo color, FD&C red #3. In collaboration with the University of New Orleans FD&C #3 food color analogs were studied (in vitro) by the ARS scientists and found to act synergistically to kill fungi (Aspergillus flavus and Fusarium graminearum). Results from this research were used to develop and implement experiments in the new in-house project to determine visib blue light effectiveness in preventing microbial spoilage of foods. Lon term, this research is expected to have wide range applicability in the protection of post harvest food from microbial spoilage. In particular, this research could have utility to improve the safety, supply, and marketability for sugar producers, citrus companies, aquaculture, and po harvest corn.
Impacts
(N/A)
Publications
- De Lucca II, A.J., Heden, L.-O., Ingber, B.F., Bhatnagar, D. 2011. Antifungal properties of wheat histones (H1-H4) and purified wheat histone H1. Journal of Agricultural and Food Chemistry. 59:6933-6939.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report 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. 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. This report documents progress for the parent Project 6435-41000-101-00D Discovery of Antifungal Compounds from Low Value/Underutilized Crops And Crop Co-Products that started in October 2009 and continues research from Project 6345-41000-097-OOD of the same title. Research on the fungicidal properties of certain soybean volatile compounds, e.g., trans-2-hexenal and trans-2-heptenal, was expanded. Our data showed that, in the volatile state, trans-2-hexenal was superior to trans-2-heptenal volatiles, in preventing Aspergillus flavus growth and aflatoxin (a potent fungally produced carcinogen on crops) production. Even after the pumped trans-2-hexenal ceases to flow, the corn displayed no Aspergillus flavus growth, suggesting that the fungus was killed, not simply inhibited from growing. Under a Confidentiality Agreement with our stakeholder, Symrise (producer of bisabolol and dragosantol), we expanded our studies of the antimicrobial spectrum of activity of these two compounds known as terpenes. Bisabolol is found in plant oils (e.g., chamomile oil), while dragosantol is the synthetic version of bisabolol. Both are antimicrobial, but are safe for human use. Our attempts to volatilize both compounds for studies in our wet corn/Aspergillus flavus assays (see above) were only partially successful. In future work, we need to develop a better protocol for volatilizing these compounds. We continued our non-funded research, begun in FY-2009, on the antimicrobial properties of wheat histones (proteins associated with deoxyribonucleic acid) provided to us by our collaborator (University of Lund, Sweden). We found that these histones are highly active against many, but not all fungi. The collaborator�s data suggests that the membrane composition of different species is most likely due to the different sensitivity we see for different microbial genera. In recent years, the scientific literature has described the use of visible light to kill microorganisms. We began exploratory experiments to determine whether blue light (470nm) could kill several different harmful bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus atrophaeus, Leuconostoc mesenteroides) and fungi (Aspergillus flavus, Fusarium verticillioides). The light, alone, kills all the bacteria, but not the fungi. However, blue light in combination with photosensitizing compounds kill fungi. Our research on determining the secondary metabolic volatiles produced only by toxigenic strains of Aspergillus flavus continues. During FY2010, we reported to our stakeholder, Sensor Development Corporation, the identities of such compounds. Based on our data, the company has developed a �second generation� prototype real-time electronic sensor that will soon undergo Beta testing in grain silos. Accomplishments 01 Identification of Secondary Metabolic Volatile Compounds Unique for Toxigenic Aspergillus flavus Grown on Corn. Stored corn can be contaminated by aflatoxin, a cancer-causing compound produced by Aspergillus flavus, when corn becomes wet. Aflatoxin is dangerous to humans and animals and, each year, costs United States Agriculture many millions of dollars in contaminated corn and other oilseeds. Current detection of this toxin requires extensive, time-consuming testing of co in a laboratory. A real-time sensor, dedicated to detect Aspergillus flavus growth, would immediately warn grain handlers of contaminated gra and reduce the need for aflatoxin testing in laboratories. Agricultural Research Service researchers (Food and Feed Safety Research Unit, Southe Regional Research Center, New Orleans, LA) successfully determined the identity of secondary metabolic volatiles specific for aflatoxin-produci strains of Aspergillus flavus when grown on sterile, wet cracked corn. Our stakeholder, Sensor Development Corporation, has used our data to develop a �second-generation� prototype sensor using proprietary electronic chips and mathematical algorithm soon to undergo Beta testing in grain silos. The impact is improved prototype sensor for the detecti of aflatoxigenic Aspergillus flavus growing in corn.
Impacts
(N/A)
Publications
- Rajasekaran, K., De Lucca II, A.J., Cary, J.W. 2009. Aflatoxin Control Through Transgenic Approaches. Toxins Reviews. 28(2):89-101.
- Cornelius, M.L., Lyn, M.E., Williams, K.R., Lovisa, M.P., De Lucca Ii, A.J. , Lax, A.R. (2009) The Efficacy of Bait Supplements for Improving the Rate of Discovery of Bait Stations in the Field by the Formosan Subterranean Termite (Isoptera: Rhinotermitidae). Journal of Economic Entomology. 102(3) :1175-1181.
- Le Blanc, B.W., Davis, O.K., Boue, S., Delucca, A., Deeby, T.A. 2009. Antioxidant Activity of Sonoran Desert Bee Pollen. Journal of Agricultural and Food Chemistry. 115, 1299-1305.
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