L. Miche et J. Balandreau, Effects of rice seed surface sterilization with hypochlorite on inoculatedBurkholderia vietnamiensis, APPL ENVIR, 67(7), 2001, pp. 3046-3052
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.