Desiccation induces viable but Non-Culturable cells in Sinorhizobium meliloti 1021

The survival of S. meliloti 1021 was estimated during nine weeks at 22% relative humidity. We found that after an initial rapid decline of colony forming units, the decline slowed to a steady 10-fold reduction in colony forming units every 22 days. In spite of the reduction in colony forming units, the fraction of the population identified as viable (42-54%) based on the Baclight live/dead stain did not change significantly over time. This change in the ability of viable cells to form colonies shows (i) an underestimation of the survival of rhizobial cells using plating methods, and that (ii) in a part of the population desiccation induces a Viable But Non Culturable (VBNC)-like state, which has not been reported before. Resuscitation attempts did not lead to a higher recovery of colony forming units indicating the VBNC state is stable under the conditions tested. This observation has important consequences for the use of rhizobia. Finding methods to resuscitate this fraction may increase the quality of powder-based seed inocula.Rhizobia form root nodules in symbiosis with legumes in which they fix atmospheric nitrogen and supply the fixed nitrogen to the plants (Jones et al. 2007). This system can be used to replenish soils with biologically-fixed nitrogen, and reduces the need for chemical fertilizers and pollution. However, this process is affected by salinity, drought and desiccation stress (Zahran 1999; Vriezen et al. 2007). To make optimal use of this system, inoculants are employed which allow for close contact between the microorganisms and the germinating seed (Smith 1992; Kosanke et al. 1999; Deaker et al. 2004). Many inoculants are powder-based, and a drying step during production reduces their quality (Kosanke et al. 1992). According to Catroux et al. (2001) many inoculants remain unreliable because of the inability of bacterial cells to persist under adverse conditions, including desiccation.Many conditions have been identified that affect the survival