Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 3, 2003
Publication Date: June 1, 2003
Citation: Bonde, M.R., Nester, S.E., Schaad, N.W., Frederick, R.D., and Luster, D.G. Improved detection of Tilletia indica teliospores in seed or soil by elimination of contaminating microorganisms with acidic electrolylized water. PLANT DISEASE. 87:712-718. 2003. Interpretive Summary: Karnal bunt is a disease that affects the ability of the United States to sell wheat to foreign countries. Even a single propagule of the fungus, if found in a wheat shipment, can cause a foreign country to reject the shipment, resulting in a huge monetary loss to the U.S. exporter. However, other fungi produce propagules that look like Karnal bunt, and if found can cause unjustified rejection by the importing country. These Karnal bunt "look-a-likes" are far more common than the real pathogen, and are a threat to U.S. wheat sales. In order to determine if an interception is Karnal bunt, or just a look-a-like, the propagule must be separated in pure form from the wheat and all other microorganisms and grown on a growth medium. It then can be tested by molecular techniques to determine true identity. We have developed a technique which greatly increases our ability to separate the pathogen in pure form from wheat and other microorganisms, and get it to grow quickly. The increased ability to make a rapid and correct identification should save large amounts of money for the U.S. wheat industry.
Technical Abstract: Acidic electrolyzed water (AEW) is a germicidal product of electrolysis of a dilute solution of sodium chloride, which can be used to disinfect wheat seed or soil samples being tested for Karnal bunt teliospores without risk of damaging the spores. The AEW we produced for our study had a pH of 2.5 to 2.8 and oxidation/reduction potential (ORP) of approximately 1130 mV. In simulations of routine extractions of wheat seeds to detect teliospores of Tilletia indica, causal agent of Karnal bunt of wheat, the effectiveness of a 30-min AEW treatment was compared to a 2-min 0.4% sodium hypochlorite (NaOCl) treatment to eradicate bacteria and non-smut fungi. Each treatment reduced bacterial and fungal populations in wheat seed extracts by 6 to 7 log10 units when determined on 2% water agar with antibiotics. Reductions of 5 log10 units or more were observed on other media. NaOCl and AEW also were very effective at eliminating bacteria and fungi from soil extracts. In studies to detect and quantitate T. indica teliospores in soil, AEW was nearly 100% effective at eliminating all non-smut organisms. The low free chlorine levels in AEW, as evidenced in this study, suggested compounds other than those with chlorine play a significant role in the sanitation effect of AEW. The low pH of AEW was shown to contribute substantially to the effectiveness of AEW to reduce microorganisms. A standardized protocol is provided, for a 30-min AEW treatment of wheat seed washes or soil extracts, to eliminate contaminating non bunt microorganisms. This protocol facilitates detection and enumeration of viable teliospores of T. indica in wheat seed or soil and the isolation of pure cultures for identification by PCR. The germicidal effects of AEW, as demonstrated in this study, illustrate the potential of AEW as an alternative to presently used pesticides in agriculture.