The time course of initiation and development of root nodules was investigated in the South American actinorhizal shrub Discaria trinervis (Rhamnaceae). A local strain of Frankia (BCU110501) which was isolated from D. trinervis nodules, was used as inoculum. Inoculated seedlings were periodically studied under the light microscope after clearing with aqueous NaClO. In parallel, semithin and ultrathin sections were analysed by light and electron microscopy. Infection by Frankia BCU110501 involved intercellular penetration among epidermal and cortical root cells. Nodule primordia were detected from 6 d after inoculation, while bacteria were progressing through intercellular spaces of the outer layers of cortical cells. Invasion of host cells by the symbiont occurred 7–9 d after inoculation, and hypertrophy of the primordium cells was associated with Frankia penetration. Root hairs were not deformed during the early events of nodule formation. From 13 to 16 d after inoculation, the proximal cellular zone of the primordia behaved differently from the other tissues after NaClO treatment and remained darkly pigmented. At the same time, differentiation of Frankia vesicles started to occur inside already infected cells. By 16 d after inoculation, spherical vesicles of BCU110501 were homogeneously distributed in the host cells. These vesicles were septate and surrounded by void space. Frankia spores or sporangia were not observed in the nodule tissue. This study has clarified the mode of Frankia penetration in D. trinervis, one of the Rhamnaceae which also includes Ceanothus. The events involved in infection, nodule induction, host-cell infection and vesicle differentiation have been characterized and identified as time-segregated developmental processes in the ontogeny of D. trinervis root nodules.

A split-root growth system was used to study photosynthate partitioning to developing nodules and roots of soybean (Glycine max L., Merr.). Opposite sides of the root systems were inoculated with Bradyrhizobium japonicum at 8 and 12 d after planting (early/delayed inoculation treatment) or, alternatively, only one side was inoculated 8 d after planting (early/uninoculated treatment). Plants were incubated with 14CO2 at 24-h intervals from early inoculation until the onset of N2 fixation (acetylene reduction). After staining with Eriochrome black, root and nodule meristematic structures were excised under a dissecting microscope and their radioactivity determined by scintillation counting. The specific radioactivity of nodule structures increased with nodule development, and was as much as 4 times higher in early nodules than in roots and nodules on half-roots receiving delayed inoculation. By the time that N2 fixation could be measured in the first mature nodules, the early inoculated half-root contained over 70% of the radioactivity recovered from the entire root systems of both early/delayed and early/uninoculated treatments. These results suggest that developing nodules create a strong sink for photosynthate, and that nodules and roots compete for current photosynthate. Early initiated nodules might develop at the expense of late initiated nodules, as well as at the expense of the roots themselves.