The unique root-nodule symbiosis between Rhizobium and the aquatic legume,Neptunia natans (L. f.) Druce

We examined the development of the aquatic N₂-fixing symbiosis between Rhizobium sp. (itNeptunia) and roots of Neptunia natans L. f. (Druce) (previously N. oleracea Lour.) under natural and laboratory conditions. When grown in its native marsh habitat, this unusual aquatic legume does not develop root hairs, the primary sites of rhizobial infection for most temperate legumes. Under natural conditions, the aquatic plant floats and develops nitrogen-fixing nodules at emergence of lateral roots on the primary root and on adventitious roots at stem nodes, but not from the stem itself. Cytological studies using various microscopies revealed that the mode of root infection involved an intercellular route of entry followed by an intracellular route of dissemination within nodule cells. After colonizing the root surface, the bacteria entered the primary root cortex through natural wounds caused by splitting of the epidermis and emergence of young lateral roots, and then stimulated early development of nodules at the base of such roots. The bacteria entered the nodule through pockets between separated host cells, then spread deeper in the nodule through a narrower intercellular route, and eventually evoked the formation of infection threads that penetrated host cells and spread throughout the nodule tissue. Bacteria were released from infection droplets at unwalled ends of infection threads, became enveloped by peribacteroid membranes, and transformed into enlarged bacteroids within symbiosomes. In older nodules, the bacteria within symbiosomes were embedded in an unusual, extensive fibrillar matrix. Cross-inoculation tests of 18 isolates of rhizobia from nodules of N. natans revealed a host specificity enabling effective nodulation of this aquatic legume, with lesser affinity for Medicago sativa and Ornithopus sp., and an inability to nodulate several other crop legume species. Acetylene reduction (N₂ fixation) activity was detected in nodules of N. natans growing in aquatic habitats under natural conditions in Southern India. These studies indicate that a specific group of Rhizobium sp. (Neptunia) occupies a unique ecological niche in aquatic environments by entering into a N₂-fixing root-nodule symbiosis with Neptunia natans.We thank J. Whallon for technical assistance, G. Truchet, J. Vasse, S. Wagener, J. Beaman, F. DeBruijn, F. Ewers, and A. Squartini for helpful comments, and N.N. Prasad and G. Birla for assistance in conducting field observations. This work was supported by the Michigan Agricultural Experiment Station and National Science Foundation grants DIR-8809640 and BIR-9120006 awarded to the MSU Center for Microbial Ecology. This study is dedicated to the memory of Dr. Joseph C. Burton, a friend and colleague who made many contributions to the study of the Rhizobiumlegume symbiosis.