Effects of rice seed surface sterilization with hypochlorite on inoculatedBurkholderia vietnamiensis

When a combination of hydrogen peroxide and hypochlorite was used to surfac e sterilize rice seeds, a 10(2)- to 10(4)-fotd decrease in CFU was observed during the first 15 h after inoculation of the rice rhizosphere organism B urkholderia vietnamiensis TVV75. This artifact could not be eliminated simp ly by rinsing the seeds, even thoroughly, with sterile distilled water. Whe n growth resumed, a significant increase in the frequency of rifampin- and nalidixic acid-resistant mutants in the population was observed compared to the control without seeds. This phenomenon was a specific effect of hypoch lorite: it was not observed with hydrogen peroxide alone. It was also not o bserved when the effect of hypochlorite was counteracted by sodium thiosulf ate. We hypothesized that the hypochlorite used for disinfection reacted wi th the rice seed surface, forming a chlorine cover which was not removed by rinsing and generated mutagenic chloramines. We studied a set of rifampin- and nalidixic acid-resistant mutants obtained after seed surface steriliza tion, The corresponding rpoB and gyrA genes were amplified and sequenced to characterize the induced mutations. The mutations in five of seven nalidix ic acid-resistant mutants and all of the rifampin-resistant mutants studied were found to correspond to single amino acid substitutions. Hypochlorite surface sterilization can thus be a source of artifacts when the initial ba cterial colonization of a plant is studied.