Contribution of plasmids to the survival of clover rhizobia [Rhizobium leguminosarum bv. trifolii] in soil and in the rhizosphere of clover

Abstract

Strains of clover rhizobia [Rhizobium leguminosarum biovar trifolii] used to inoculate seeds of arrowleaf clover [Trifolium vesiculum Savi] often fail to survive in the field. The contribution of plasmids to the saprophytic competence of clover Rhizobium W14-2, a strain well adapted to survival under adverse soil conditions, was investigated in the laboratory. The four plasmids in W14- 2 were sequentially labelled with transposon Tn5-Mob-sac and eliminated (cured) by a heat treatment The levansucrase-coding sac genes render rhizobia sensitive to sucrose and plasmid-cured derivatives were selected on sucrose-containing medium. The wild-type and its plasmid-cured derivatives were then compared in vitro and in sterilized soil under laboratory conditions. Derivatives with all possible combinations of plasmids but one combination (plasmids a and d together) were obtained. The presence of plasmids influenced the performance in vitro of strain W14-2, its survival in soil under heat or drought stress, and its growth in soil and in the rhizosphere of clover. Plasmid a contributed to growth in vitro and in soil, mainly by reducing the length of the lag phase. Plasmid a also contributed to the competitiveness of W14-2 in the rhizosphere of clover, an effect that may be linked to its positive role in cell motility. Both plasmids b and c contributed to growth in several defined and complex media, in soil, and in the rhizosphere of clover, as well as to survival under heat stress in soil. Plasmid c was also beneficial under drought stress. Plasmid d, the symbiotic plasmid, increased the length of the lag phase in Hynes-Puhler broth and was detrimental under drought stress. The role of plasmid d in the other phenotypes was essentially neutral. Results indicate that plasmids are important to the autecology of rhizobia in soil and in the rhizosphere of the legume host, and that the full plasmid content contributes to the saprophytic competence of W 14-2.