Effects of Rice Seed Surface Sterilization with Hypochlorite On Inoculated Burkholderia Vietnamiensis

Effects of Rice Seed Surface Sterilization with Hypochlorite on Inoculated Burkholderia vietnamiensis

Lucie Miché and Jacques Balandreau,Laboratoire d’écologie Microbienne, UMR 5557 CNRS-Université Claude Bernard Lyon 1, 69622 Villeurbanne cedex, France , Received 31 January 2001/Accepted 23 April 2001

When a combination of hydrogen peroxide and hypochlorite was used to surface sterilize rice seeds, a 10²– to 10⁴-fold decrease in CFU was observed during the first 15 h afterinoculation of the rice rhizosphere organism Burkholderia vietnamiensisTVV75. This artifact could not be eliminated simply by rinsing the seeds, even thoroughly, with sterile distilled water. When 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 hypochlorite; it was not observed with hydrogen peroxide alone. It was also not observed when the effect of hypochloritewas counteracted by sodium thiosulfate. We hypothesized that the hypochlorite used for disinfection reacted with 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 sterilization. The corresponding rpoB and gyrA genes were amplified and sequenced to characterize the induced mutations. The mutations in five of seven nalidixic acid-resistant mutants and all of the rifampin-resistant mutants studied were found to correspond tosingle amino acid substitutions. Hypochlorite surface sterilizationcan thus be a source of artifacts when the initial bacterial colonizationof a plant is studied.