2012 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.
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.
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, Fusarium (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 coli, Bacillus (B.) subtilis, B. atrophaeus) activity below 20µM 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 our storage model and (2) antimicrobial food grade films.
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) produce 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 to study antimicrobials targeting B. anthracis, the microbe causing anthrax. Alone, blue light was not found to be fungicidal, though fungal viability was reduced in the presence of photosensitizing compounds such as the food 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 visible blue light effectiveness in preventing microbial spoilage of foods. Long-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 post harvest corn.
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 the development of a sensor by our Stakeholder, Sensor Development Corporation. 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 use.
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.