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United States Department of Agriculture

Agricultural Research Service

Research Project: NOVEL MICROBIAL SENSING AND ELIMINATION TECHNOLOGIES FOR PROTECTION OF AGRICULTURAL COMMODITIES
2013 Annual Report


1a.Objectives (from AD-416):
1. Detection and control of fungi in post-harvest, stored corn. 2. Evaluate commercially available plant compounds for their in vitro activity against agriculturally important bacteria and fungi.


1b.Approach (from AD-416):
This project investigates new technologies to detect Aspergillus (A.) flavus and Fusarium (F.) verticillioides growth on corn and novel methods to inhibit their growth and mycotoxin production. Secondary metabolic volatiles unique for the growth of toxigenic strains of the fungi will be identified and shared with our stakeholder who will develop electronic, “real-time” sensors to detect the growth of these fungi. Safe, commercially available plant antifungal volatiles will be tested for their in vitro activity against these fungi. Promising compounds will be further tested to determine their ability to prevent fungal growth in small-scale, bench models of stored corn. These same compounds in the liquid form will be studied for their in vitro activity against the bacterium, Leuconostoc (L.) mesenteroides (a problem in sugar cane factories) and the fungi Penicillium (P.) digitatum and P. italicum which cause post harvest citrus rot. ARS collaborators will use this data to develop novel technologies to prevent the growth of these fungi in raw sugar cane juice and stored oranges.


3.Progress Report:
This serves as a final report for project 6435-41000-107-00D. Progress was made on all proposed research in the new project plan. Research was carried out on the identification of unique volatiles (compounds with ability to evaporate) produced by toxigenic strains of Fusarium (F.) verticllioides as compared to non-toxigenic strains and corn controls, when grown separately on sterile and non-sterile corn. Initial results from separate multiple runs using one strain each of toxigenic and one atoxigenic F. verticllioides indicate that unique volatiles are produced by the former. This initial study was followed with studies using non-sterile, wet corn as the growth substrate for the same fungal isolates. In order to identify safe, inexpensive, commercially available antimicrobials, the following were used in in vitro assays: (1) volatile compounds to protect stored corn from the growth of mycotoxin producing fungi, (2) non-volatile compounds to add to food grade protective films, and (3) water soluble compounds to prevent Leuconsotoc mesenteroides (a bacteria) growth. This initial step in our program continued during FY 2013. During early FY 2013, research was completed on the ability of intermittently pumped, volatilized trans-2-hexenal (T-2-H), a chemical compound, to prevent bacterial and fungal growth on non-sterile wet corn in a bench-top model of stored corn. Data showed that intermittent pumping (30 min every 2 hr) prevented bacterial and fungal growth from time zero. Pumping (30 min every 12 hr) prevented all fungal growth from time zero but only killed bacteria sometime between 24 and 72 hrs after pumping commenced. Also during FY 2013, research continued on the ability of blue light (peak: 470 nm) to prevent Aspergillus (A.) flavus fungal growth inoculated onto non-sterile corn. The initial study, using a single layer of corn in a Petri dish, clearly showed that growth of inoculated A. flavus as well as the naturally-occurring fungi was prevented by blue light treatment. Next we began studies of 8 oz of corn being turned in a glass cylinder between two banks of blue light to determine whether the light can prevent fungal growth on multiple layers of corn. At this time we found that the lights had lost 50% of their energy (mW) and that this level was insufficient to prevent growth.


4.Accomplishments
1. Detection of unique volatiles produced by Fusarium (F.) verticillioides could be used to remove contaminated corn from storage and thereby reduce the threat of Fusarium species mycotoxins from food and feed. Under certain conditions of moisture and temperature, the naturally present mycotoxin fungi (e.g., Aspergillus (A.) and Fusarium spp.) can grow on stored corn and render it unsafe. After successfully completing a study of A. flavus produced volatiles unique to toxigenic strains, ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, in FY 2013, began a multi-year study to identify novel volatiles produced by toxigenic strains of F. verticllioides. Studies progressed with sterile corn as a growth substrate. After several toxigenic and atoxigenic isolates were studied and the unique volatiles for the toxigenic isolates determined, similar experiments were performed with non-sterile corn. Our stakeholder, Sensor Development Corporation, used data from a previously completed project to develop an electronic sensor for volatiles unique to aflatoxin-producing A. flavus. A similar sensor could be constructed using data obtained from this study with F. verticllioides upon completion.

2. Safe, effective, and inexpensive plant volatiles prevent microbial, especially toxigenic fungal, growth and toxin production in a stored corn model. In FY 2011-12, ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, completed a study that showed volatilized trans-2-hexenal (T-2-H) was highly effective against Aspergillus flavus both in vitro and when applied to wet, sterile corn in small models of stored corn.

3. In vitro research on the antifungal and antibacterial properties of safe, commercially available plant compounds to determine which are suitable candidates to protect post-harvest food and feed. Work was completed by ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, on the antifungal properties of three such compounds (limonene, carvacrol, bisabolol, dragosantol, pinene, linalool) against the non-germinated (NG) and germinating (G) conidia (equivalent of seed in plants) of Fusarium (F.) verticillioides, Aspergillus (A.) niger, Penicillium (P.) digitatum, and P. italicum). The NG and G conidia of F. verticillioides and P. italicum were resistant to dragosantol and bisabolol at = 20 µM. The NG conidia of A. niger were significantly susceptible to carvacrol (15 µM) and linalool (20 µM) while the G conidia were significantly susceptible at 5 µM for both compounds. Pinene and limonene cause significant mortality to NG and G conidia of F. verticillioides beginning at 5 µM. Bioassays defined the antimicrobial activity of eugenol and vanillin against a panel of bacteria and toxigenic fungi having agricultural importance.

4. Visible blue light (470 nm) is antimicrobial with potential use in food protection. Ultraviolet light (wavelength 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, performed initial studies that showed this wavelength prevented fungal growth, both naturally occurring and inoculated Aspergillus (A.) flavus, on single layers of non-sterile, wet corn. Next, initial studies were performed using 8 oz of similar corn being turned in a glass cylinder with two blue light arrays in parallel on the outside. Lack of antifungal activity lead to a check of the light (LED) energy (mW) level which showed the output was about 50% lower than specifications.


Last Modified: 12/18/2014
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