Abstract: Polymerase chain reaction(PCR) assays are quicker and often just as sensitive as agar plating assays for detecting plant pathogenic bacteria. However, PCR can be used to detects both live and dead cells. Since dead cells do not cause disease, their detection can be a problem in seed-health testing. Ethidium monoazide(EMA), a DNA binding dye, can prevent the amplification of DNA from dead cells of food-borne bacteria. To test the use of EMA for detecting live cells of Acidovorax avenae subsp. citrulli(Aac), seed extracts, spiked with Aac containing 3×10⁶, 3×10⁷, and 3×10⁸ cfu/mL, were treated with EMA at 0, 1, 2, 3, and 4 mg/L and incubated at room temperature and at 75 ℃ for 3 min to kill Aac. After 10 min in the dark, the mixtures were put on ice under light for 5, 10, and 15 min to allow EMA to be effective, then assayed by classical PCR and agar plating. The results show that heat-treated suspensions containing dead cells of Aac at a level of 3×10⁶ cfu/mL were PCR negative when pretreated with 2 mg/L or greater EMA and 10 min or more exposures. However, all treatments without EMA containing Aac at 3×10⁶ cfu/mL or greater were ineffective as PCR results were positive. These preliminary results show that the addition of EMA to seed extracts containing moderate numbers of Aac has a potential for the specific detection of live cells.

Key words: Ethidium monoazide, Interaction, Acidovorax avenae subsp. citrulli, PCR, Live-dead cell