Enhanced nodule initiation on alfalfa by wild-type Rhizobium meliloti co-inoculated with nod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-type Rhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functional nodA, nodC, nodE, nodF or nodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation with R. trifolii or Agrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by the R. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid from R. meliloti into A. tumefaciens abolished this stimulation. Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant of R. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-type R. meliloti. These results indicate that certain nod gene mutants and members of the Rhizobiaceae may produce extracellular "signals" that supplement the ability of wild-type R. meliloti cells to induce crucial responses in the host.