Influence of water potential on the enumeration of Bradyrhizobium sp (cicer) by Tn5 mutants (km-) and fluorescent antibody

Abstract

Tn5 induced insertion mutants were generated in Bradyrhizobium sp (Cicer) by mating with Escherichia coli MV-12 carrying the suicide plasmid pGS9. One Tn5 mutant of strain A8 was selected, inoculated in nonsterile soil at three water potentials (-0.033, -0.3 and -3 MPa) and enumerated for survival by plate count using kanamycin (Km) amended medium and by the fluorescent antibody (FA). Survival, as determined by plate count and FA was optimum at -0.3 MPa where the strain remained viable at near the initial inoculum level for at least 55 days. At the other two water potentials, bradyrhizobial viable counts decreased sharply, particularly at -3 MPa. Initially, FA and plate counts were similar, but 10 days after inoculation, estimates by FA were two log units higher than by plate count for soils held at -3 MPa. The discrepancy between the two methods may be due to identification of fluorescent active dead cells or failure of alive, but stressed cells to grow on the artificial medium. Under minimum water stress (-0.033 and -0.3 MPa water potential), results with both methods were similar. These results show that Tn5 mutagenesis can be used to mark bacteria and to conduct effective ecological studies. Additionally, they indicate that FA enumeration tends to overestimate the bradyrhizobial population under some dry soil conditions that reduce the microbial activity in soil.