Induction of the viable but nonculturable state of Ralstonia solanacearum by low temperature in the soil microcosm and its resuscitation by catalase

PLoS One. 2014 Oct 8;9(10):e109792. doi: 10.1371/journal.pone.0109792. eCollection 2014.

Abstract

Ralstonia solanacearum is the causal agent of bacterial wilt on a wide variety of plants, and enters a viable but nonculturable (VBNC) state under stress conditions in soil and water. Here, we adopted an artificial soil microcosm (ASM) to investigate the VBNC state of R. solanacearum induced by low temperature. The culturability of R. solanacearum strains SL341 and GMI1000 rapidly decreased at 4°C in modified ASM (mASM), while it was stably maintained at 25°C in mASM. We hypothesized that bacterial cells at 4°C in mASM are viable but nonculturable. Total protein profiles of SL341 cells at 4°C in mASM did not differ from those of SL341 culturable cells at 25°C in mASM. Moreover, the VBNC cells maintained in the mASM retained respiration activity. Catalase treatment effectively restored the culturability of nonculturable cells in mASM, while temperature increase or other treatments used for resuscitation of other bacteria were not effective. The resuscitated R. solanacearum from VBNC state displayed normal level of bacterial virulence on tomato plants compared with its original culturable bacteria. Expression of omp, oxyR, rpoS, dps, and the 16S rRNA gene quantified by RT-qPCR did not differ significantly between the culturable and VBNC states of R. solanacearum. Our results suggested that the VBNC bacterial cells in mASM induced by low temperature exist in a physiologically unique state.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalase / metabolism*
  • Culture Techniques
  • Gene Expression Regulation, Bacterial
  • Microbial Viability*
  • Ralstonia solanacearum / genetics
  • Ralstonia solanacearum / growth & development
  • Ralstonia solanacearum / physiology*
  • Soil*
  • Stress, Physiological
  • Temperature*

Substances

  • Soil
  • Catalase

Grants and funding

This research was supported by the Mid-career Researcher Program through the National Research Foundation of Korea grant funded by the Ministry of Education, Science and Technology (NRF-2012R1A2A2A01045039) and by a grant from the Next-Generation BioGreen 21 Program (No. PJ0082012014), Rural Development Administration, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.