Association of Rhizobium Strains with Roots of Trifolium repens

TWO TECHNIQUES WERE USED TO ASSESS THE BINDING OF RHIZOBIA TO CLOVER ROOTS: indirect counting after radiolabeling the bacteria and direct counting by using phase-contrast microscopy. Microscopic observations revealed a large variability in the number of bacteria associated with individual root hairs. This variability made unbiased counting by microscopy difficult. Systematic examination of all visible root hairs and "blind" counting of coded strains and treatments were adopted to minimize observer bias. The validity of the radiolabeling method was also examined in some detail. The reproducibility of results from this method was satisfactory. However, drawbacks of this method included its lack of sensitivity and its failure to distinguish between bacteria attached to mature root hairs, emerging root hairs, and undifferentiated epidermal cells. The method also failed to distinguish between individual bacteria and any aggregates that may be present. The ability of a number of chosen mutant strains of Rhizobium trifolii and their corresponding parent strains, as well as a number of nonhomologous strains, to bind to clover roots was assessed by using both of these methods. Our results gave no indication of specificity of R. trifolii binding to clover roots. 2-Deoxy-d-glucose did not appear to have a major inhibitory effect on the attachment of rhizobia to the host root, which suggests that lectin cross-bridging is not an obligatory step in the initiation of infection even though it may occur under some conditions. The presence or absence of the symbiotic plasmid was not correlated with bacterial adherence to the host plant root. Since host specificity functions are carried on this plasmid, our results suggest that binding of rhizobia to the legume root is not the basis of host specificity.