Time course of nodule development in the Discaria trinervis (Rhamnaceae) Frankia symbiosis

The time course of initiation and development of root nodules was investiga ted in the South American actinorhizal shrub Discaria trinervis (Rhamnaceae ). A local strain of Frankia (BCU1 10501) which was isolated from D, triner vis nodules, was used as inoculum. Inoculated seedlings were periodically s tudied under the light microscope after clearing with aqueous NaClO. In par allel, semithin and ultrathin sections were analysed by light and electron microscopy. Infection by Frankia BCU110501 involved intercellular penetrati on among epidermal and cortical root cells. Nodule primordia were detected from 6 d after inoculation, while bacteria were progressing through interce llular spaces of the outer layers of cortical cells. Invasion of host sells by the symbiont occurred 7-9 d after inoculation, and hypertrophy of the p rimordium cells was associated with Frankia penetration. Root hairs were no t deformed during the early events of nodule formation. From 13 to 16 d aft er inoculation, the proximal cellular zone of the primordia behaved differe ntly from the other tissues after NaClO treatment and remained darkly-pigme nted. At the same time, differentiation of Frankia vesicles started to occu r inside already infected cells. Bq 16 d after inoculation, spherical resid es of BCU110501 were homogeneously distributed in the host cells. These ves icles were septate and surrounded by void space. Frankia spores or sporangi a were not observed ed in the nodule tissue. This study has clarified the m ode of Frankia penetration in D, trinervis, one of the Rhamnaceae which als o includes Ceanothus. The events involved in infection, nodule induction, h ost-cell infection and vesicle differentiation have been characterized and identified as time-segregated developmental processes in the ontogeny of D. trinervis root nodules.