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

The objective of this study was to determine the efficacy of acidic electrolyzed water (AEW) to eliminate microorganisms other than Tilletia indica Mitra teliospores from wheat seed and soil extracts. The teliospores then can be identified by a polymerase chain reaction (PCR) test (9,10,26). Growth of contaminants can obscure germinating T. indica teliospores and inhibit teliospore germination (M. R. Bonde and S. E. Nester, personal observation). The AEW treatment is part of a proven protocol to detect T. indica teliospores in wheat seed samples using a bioPCR based technique (23). However, the level of germicidal effectiveness of AEW for reducing contaminating microorganisms in wheat (or soil) extracts when testing for T. indica has not been reported. Karnal bunt of wheat first was discovered in India in 1930 (20) and since has been found in Pakistan, Nepal, Iraq, Afghanistan (5), northwest Mexico (8), southwest United States (34), and South Africa (7). Although Karnal bunt usually causes only minor yield and quality reductions (1,11), it is the subject of strict international quarantines (5). Teliospores of T. indica detected in a wheat shipment can cause rejection of the consignment by certain countries. As a result, USDA initiated a Karnal bunt research program in 1982 (5,25). Immediately following the discovery of Karnal bunt in Arizona and California in 1996 (34), extensive field surveys were conducted in those states, and a national Karnal bunt survey was initiated to determine the extent to which Karnal bunt might be present in harvested grain in other areas of the country. Differentiation of T. indica from morphologically similar species sometimes required a method other than microscopy (5). For example, Tilletia walkeri from rye grass, and T. horrida from rice, have teliospores that are morphologically similar to those of T. indica, which sometimes are present as contaminants in harvested wheat grain (6). The method we used for species determination was based upon the PCR using T. indica species-specific DNA primers, and required germination of suspect teliospores to obtain sufficient DNA for the test (10). As part of the original protocol to germinate teliospores, the extract that was obtained by size-selective sieving of grain samples was treated for 2 min with 0.4% sodium hypochlorite (NaOCl) (21). The NaOCl treatment eliminated most other contaminating microorganisms whose DNA could interfere with PCR assays, and stimulated teliospore germination (3). However, a major drawback was that bunt teliospores sometimes would not germinate due to dormancy or possibly due to damage by the harsh NaOCl treatment. Lack of germination could retard timely identifications. At the time of the National Karnal Bunt Survey in 1996 and 1997, the United States Department of Agriculture-Agricultural Research Service (USDA-ARS) at Fort Detrick was testing AEW as a means of decontaminating harvested grain potentially contaminated with T. indica teliospores. AEW has been referred to by many names, including superoxidized water (24), electrolyzed saline solution (32), and functional water (17). Most research on its sanitizing capabilities has been conducted with bacteria (14,24,28–30). In medical practice in Japan, AEW is used to treat mediastinitis infection after cardiovascular surgery (13). AEW is a product of electrolysis of a dilute solution of sodium chloride and contains a low concentration of hypochlorous acid, which is a very effective sanitizer at low pH (31). In preliminary tests in our laboratory, AEW treatments of 20 to 30 min killed bacteria and fungi, including Aspergillus, Cladosporium, and Penicillium spp., while stimulating germination of teliospores of T. indica (4). However, at ABSTRACT Bonde, M. R., Nester, S. E., Schaad, N. W., Frederick, R. D., and Luster, D. G. 2003. Improved detection of Tilletia indica teliospores in seed or soil by elimination of contaminating microorganisms with acidic electrolyzed water. Plant Dis. 87:712-718.