Effect of trehalose on survival of Bradyrhizobium japonicum during desiccation

Abstract

Aims: A major reason for the ineffectiveness of legume inoculants in the field is the rapid death of rhizobia because of desiccation. The major purpose of this study was to identify conditions under which a,a-trehalose would improve survival of Bradyrhizobium japonicum during desiccation. Methods and Results: Trehalose was added to cultures just prior to desiccation or was supplied to bacteria during the 6-day growth period. A wide variety of trehalose concentrations was tested. Trehalose added to cultures at the time of desiccation improved survival slightly, but trehalose loading during growth was much more effective in protection against desiccation. Growth of bacteria with 3 mmol l )1 trehalose increased trehalose concentration in cells by about threefold and increased survival of cells placed on soya bean [Glycine max (L.) Merr.] seeds by two-to four-fold after 2 or 24 h. Average of overall results indicate that growth of bacteria with trehalose in the medium resulted in a 294% increase in survival after 24 h of desiccation. The concentration of trehalose in cells was very highly correlated with survival of bacteria. When trehalose-loaded cells were suspended in buffer or water, 60-85% of cellular trehalose was lost in about 1 h and, in spite of these losses, survival during desiccation was not reduced. Conclusions: Accumulation of trehalose in the cytoplasm is critical to the survival of B. japonicum during desiccation. Increasing the periplasmic concentration of trehalose is also beneficial but is not so critical as the concentration of trehalose in the cytoplasm. Because B. japonicum cannot utilize trehalose as a carbon source, cells can be loaded with trehalose by providing the disaccharide during the growth period. Significance and Impact of the Study: Although it may not be practical to use trehalose as a carbon source in inoculant production, it may be possible to engineer greater trehalose accumulation in rhizobia. Trehalose concentration in cells should be a useful predictor of survival during desiccation.

The Physiological Mechanisms of Desiccation Tolerance in Rhizobia

“…Similarly, Streeter (2003) reported that cell numbers of B. japonicum USDA110 were significantly higher in cultures with trehalose added to the basal medium compared with cultures with added maltose after three to four days of growth. Although Streeter (2003) The accumulation of intracellular trehalose by B. japonicum USDA110 was increased by supplying trehalose in the growth medium.…”

“…Adding 3 mM trehalose to the growth medium of B. japonicum USDA110 increased intracellular trehalose accumulation by three-fold, which in turn resulted in a 294% increase in survival of cells after 24 hours of being air-dried on soybean seed (Streeter, 2003). Streeter (2007) reported that adding sodium chloride and trehalose to the growth medium of USDA110 cultures resulted in a larger increase in trehalose accumulation than using trehalose alone.…”

“…The addition of 3 mM trehalose to the growth medium increased intracellular trehalose concentration by threefold to 6.6 µg/mL in B. japonicum USDA110 cell culture and cells grown in 6 and 9 mM exogenous trehalose increased trehalose contents to 10.3 and 12.5 µg/mL respectively (Streeter, 2003). It was concluded that although USDA110 does not grow when trehalose is the sole source of carbon, it is able to accumulate the sugar at a slow rate in response to increased external osmotic pressure as a way of maintaining isosmotic equilibrium.…”

“…A two-to threefold increase in intracellular trehalose resulted in a 294% increase in survival of USDA110 after 24 hours desiccation on soybean seed (Streeter, 2003). There was 70% recovery of cells of NZP561 after two and a half hours of air drying when intracellular trehalose was 140 µg/mg of protein.…”

“…Trehalose is a non-reducing disaccharide which is believed to act as a compatible solute by maintaining membrane structure through the formation of biological glasses which protect proteins and other macromolecules from denaturation during desiccation (Crowe et al, 1984). The intracellular accumulation of trehalose in rhizobia has been observed to significantly improve desiccation tolerance (Streeter, 2003;McIntyre et al, 2007;Streeter, 2007).…”