Soil moisture deficit selects for desiccation tolerant Rhizobium leguminosarum bv. trifolii

Tolerance to desiccation is a highly desirable attribute for Rhizobium spp., which are widely used as symbionts of pasture legumes. Poor survival of Rhizobium spp. is of particular concern during the commercial formulation and seed application phase, but also when sown into dry fields and during saprophytic survival. Efforts have been made to protect Rhizobium spp. from desiccation, either by use of desiccation protectants in formulations, or by triggering generalised mechanisms of cellular stress tolerance. This study looked at the potential to isolate desiccation tolerant strains of Rhizobium leguminosarum bv. trifolii that are intrinsically tolerant to desiccation. We investigated the influence of long term exposure to dry soil conditions, and rapid soil drying, as factors in selection of desiccation tolerance. Pasture soils were collected from 26 sites in New Zealand that ranged in soil moisture deficits (SMD) from 149 days per year (very dry) to 0 (wet). Rhizobium leguminosarum bv. trifolii populations were quantified (most probable number; MPN) in the field-fresh and dried (48 h forced air) samples from each site, and the percentage population survival determined. Rhizobium leguminosarum bv. trifolii survival after soil drying was strongly correlated to SMD (P < 0.001; R2 = 0.42), providing evidence for strong selection for desiccation tolerant phenotypes in dry environments. Desiccation tolerance of R. leguminosarum bv. trifolii isolated from nodules taken from white clover plants grown in fresh and dried soils from three sites was compared in a micro-droplet assay. Strains from plants grown in the dried soils were more tolerant to rapid drying under in vitro conditions (P < 0.001). Screening of a subset of strains on a range of osmolytes (phenotype microarray), showed desiccation tolerance was associated with tolerance to the salts sodium nitrite, sodium nitrate, and ammonium sulphate, and ethylene glycol. Growth in the presence of ethylene glycol maybe useful surrogate indicators of desiccation tolerance in R. leguminosarum bv. trifolii. Overall, these results show that environmental conditions can provide a basis for selection of populations with desiccation tolerance, and plant-based baiting can be used to recover strains with improved characteristics.