The viable-but-nonculturable state induced by abiotic stress in the biocontrol agent Pseudomonas fluorescens CHA0 does not promote strain persistencein soil

The effects of oxygen limitation, low redox potential, and high NaCl stress for 7 days in vitro an the rifampin-resistant biocontrol inoculant Pseudom onas fluorescens CHA0-Rif and its subsequent persistence in natural soil fo r 54 days were investigated. Throughout the experiment, the strain was moni tored using total cell counts (immunofluorescence microscopy), Kogure's dir ect viable counts, and colony counts ion rifampin-containing plates), Under in vitro conditions, viable-but-nonculturable (VBNC) cells of CHAO-Rif wer e obtained when the strain was exposed to a combination of low redox potent ial (230 mV and oxygen limitation. This mimics a situation observed in the field, where VBNC cells of the strain were found in the waterlogged sail la yer above the plow pan. Here, VBNC cells were also observed in vitro when C HA0-Rif Has subjected to high NaCl levels (i.e NaCl at 1.5 M but not 0.7 M) . In all treatments, cell numbers remained close to the inoculum level for the first 12 days after inoculation of soil, regardless of the cell enumera tion method used, but decreased afterwards. At the last two samplings in so il, VBNC cells of CHA0-Rif were found in ail treatments except the one in w hich log-phase cells had been used. In the two treatments that generated hi gh numbers of VBNC cells in vitro, VBNC cells did not display enhanced pers istence compared dth culturable cells once introduced into son, which sugge sts that this VBNC state did not represent a physiological strategy to impr ove survival under adverse conditions.