Miché L, Balandreau J
Laboratoire d'Ecologie Microbienne, UMR 5557 CNRS-Université Claude Bernard Lyon 1, 69622 Villeurbanne cedex, France.
Appl Environ Microbiol. 2001 Jul;67(7):3046-52. doi: 10.1128/AEM.67.7.3046-3052.2001.
When a combination of hydrogen peroxide and hypochlorite was used to surface sterilize rice seeds, a 10(2)- to 10(4)-fold decrease in CFU was observed during the first 15 h after inoculation of the rice rhizosphere organism Burkholderia vietnamiensis TVV75. 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 hypochlorite was 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 to single amino acid substitutions. Hypochlorite surface sterilization can thus be a source of artifacts when the initial bacterial colonization of a plant is studied.
当使用过氧化氢和次氯酸盐的组合对水稻种子进行表面消毒时，在接种水稻根际微生物越南伯克霍尔德氏菌TVV75后的最初15小时内，观察到菌落形成单位（CFU）下降了10²至10⁴倍。即使使用无菌蒸馏水彻底冲洗种子，这种假象也无法简单消除。当生长恢复时，与未处理种子的对照组相比，观察到群体中耐利福平及耐萘啶酸突变体的频率显著增加。这种现象是次氯酸盐的特定作用；单独使用过氧化氢时未观察到这种现象。当用硫代硫酸钠抵消次氯酸盐的作用时，也未观察到这种现象。我们推测，用于消毒的次氯酸盐与水稻种子表面发生反应，形成一层冲洗不掉的氯覆盖层，并产生诱变氯胺。我们研究了一组种子表面消毒后获得的耐利福平及耐萘啶酸突变体。对相应的rpoB和gyrA基因进行扩增和测序，以表征诱导突变。在所研究的7个耐萘啶酸突变体中的5个以及所有耐利福平突变体中发现的突变均对应于单个氨基酸替换。因此，在研究植物的初始细菌定殖时，次氯酸盐表面消毒可能会产生假象。
