Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1-10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4′-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21-24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a "hybrid" structure intermediate between a nodule and a lateral root.