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Improved Detection of Tilletia indica Teliospores in Seed or Soil by Elimination of Contaminating Microorganisms with Acidic Electrolyzed Water

Acidic electrolyzed water (AEW) is a germicidal product of electrolysis of a dilute solution (e.g., 0.4% vol/vol) of sodium chloride. This solution can be used to disinfest wheat seed or soil samples being tested for teliospores of Tilletia indica, causal agent of Karnal bunt, without risk of damaging the teliospores. The AEW used in this study had a pH of 2.5 to 2.8 and oxidation-reduction potential of approximately 1,130 mV. In simulations of routine extractions of wheat seed to detect teliospores of T. indica, the effectiveness of a 30-min AEW treatment was compared with a 2-min 0.4% sodium hypochlorite (NaOCl) treatment to eradicate bacteria and nonsmut 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 nonsmut organisms. Free chlorine levels in AEW were very low, suggesting that compounds other than those with chlorine play a significant role in sanitation by AEW. The low pH of AEW was shown to contribute substantially to the effectiveness of AEW to reduce microorganisms. A standardized protocol is described for a 30-min AEW treatment of wheat seed washes or soil extracts to eliminate contaminating microorganisms. A significant advantage of the use of AEW over NaOCl is that, with AEW, teliospore germination is not reduced and usually is stimulated, whereas teliospore germination declines after contact with NaOCl. The 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 polymerase chain reaction. The germicidal effects of AEW, as demonstrated in this study, illustrate the potential of AEW as an alternative to presently used seed disinfestants.