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.

More than 200 angiosperms, distributed in 25 genera, develop root nodule symbioses (actinorhizas) with soil bacteria of the actinomycetous genus Frankia. Although most soils studied contain infective Frankia, cultured strains are available only after isolation from root nodules. Frankia infects roots via root hairs in some hosts or via intercellular penetration in others. The nodule originates in the pericycle. The number of nodules in Alnus is determined by the plant in an autoregulated process that, in turn, is modulated by nutrients such as nitrogen and phosphate. Except in the genera Allocausarina and Casuarina, Frankia in nodules develops so-called vesicles where nitrogenase is localized. Sporulation of Frankia occurs in some symbioses. As a group, actinorhizal plants show a large range of anatomical and biochemical adaptations in order to balance the oxygen tension near nitrogenase. In symbioses with well aerated nodule tissue like Alnus, the vesicles have a multilayered envelope composed mainly of lipids, bacterio-hopanetetrol and their derivatives. This envelope is assumed to retard the diffusion of oxygen into the nitrogenase-containing vesicle. In symbioses like Casuarina, the infected plant cells themselves, rather than Frankia, appear to retard oxygen diffusion, and high concentrations of haemoglobin indicate an infected region with a low oxygen tension. At least in Alnus spp., ammonia resulting from N2 fixation is assimilated by glutamine synthetase in the plant. The carbon compound(s) used by Frankia in nodules is not yet known. Nitrogenase activity decreases in response to a number of environmental factors but recovers upon return to normal conditions. This dynamism in nitrogenase activity is often explained by loss and recovery of active nitrogenase and has been traced to loss and recovery of the nitrogenase proteins themselves. Recovery is partly due to growth of Frankia and to development of new vesicles in the Alnus nodules. In the field, varying conditions continuously affect the plants and the measured rate of N2 fixation is a result not only of the conditions prevailing at the moment but also of the conditions experienced over preceding days. N2 fixed by actinorhizal plants is substantial and actinorhizal plants have great potential in soil reclamation and in various types of forestry. Several species are also useful in horticulture.