Abstract
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.
Similar content being viewed by others
References
Alazard, D., Ndoye, I., Dreyfus, B. (1988) Sesbania rostrata and other stem-nodulated legumes. In: Nitrogen fixation: hundred years after, pp. 765–769. Bothe, H., DeBruijn, F., Newton, W., eds. Gustav Fischer, Stuttgart
Allen, O.N., Allen, E.K. (1981) The Leguminosae. A source book of characteristics, uses, and nodulation. University of Wisconsin Press, Madison
Barrios, E., Herrara, R. (1994) Nitrogen cycling in a seasonally flooded forest: soil nitrogen mineralization and nitrification. J. Trop. Bot. 10, 399–416
Batut, J., Bostard, P., Debelle, F., Denarie, J., Ghai, J., Houget, T., Infante, D., Martinez, E., Rosseberg, C., Vasse, J., Truchet, G. (1985) Developmental biology of the Rhizobium meliloti-alfalfa symbiosis: a joint genetic and cytological approach. In: Nitrogen fixation research progress, pp. 109–115, Evans, H., Bottomley, P., Newton, W., eds. Martinus Nijhoff, Boston
Brown, S.M., Walsh, K.B. (1994) Anatomy of the legume cortex with respect to nodule permeability. Austr. J. Plant Physiol. 21, 49–68
Callaham, D., Torrey, J. (1981) The structural basis for infection of root hairs of Trifolium repens by Rhizobium. Can. J. Bot. 59, 1647–1664
Chandler, M.R. (1978) Some observations on infection of Arachis hypogaea L. by Rhizobium. J. Exp. Bot. 29, 749–755
Dazzo, F. (1982) Leguminous root nodules. In: Experimental microbial ecology, pp. 431–446, Burns, R., Slater, J., eds. Blackwell, Oxford, UK
Dazzo, F., Brill, W. (1979) Bacterial polysaccharide which binds Rhizobium trifolii to clover root hairs. J. Bacteriol. 137, 1362–1373
DeBruijn, F. (1989) The unusual symbiosis between the diazotrophic stem-nodulating bacterium Azorhizobium caulinodans ORS571 and its host, the tropical legume Sesbania rostrata. In: Plant-microbe interactions: molecular and genetic perspectives, pp. 457–504. Kosuge, T., Nester, E.., eds. McGraw-Hill, New York
De Faria, S., Hay, G., Sprent, J. (1988) Entry of rhizobia into roots of Mimosa scabrella Bentham occurs between epidermal cells. J. Gen. Microbiol. 134, 2291–2296
Dreyfus, B., Alazard, D., Dommergues, Y. (1986) Stem-nodulating rhizobia. In: Current perspectives in microbial ecology, pp. 161–169, Reddy, C., Klug, M., eds. American Society for Microbiology, Washington, D.C.
Fahraeus, G. (1957) The infection of clover roots by nodule bacteria studied by a simple glass slide technique. J. Gen. Microbiol. 16, 374–381
Goh, C.J., Lim, C.H., Lim, G., Louis, I. (1989) Nitrogen fixation by nodules of some tropical legume and non-legume plants. In: Proceedings of the Japanese Society for the Promotion of Science —National University of Singapore inter-faculty seminar, pp. 145–159, Lim, G., Katsuya, K., eds. National University of Singapore, Singapore
Hardy, R.W., Holsten, R., Jackson, E., Burns, R. (1968) The acetylene-ethylene assay for nitrogen fixation: laboratory and field evaluation. Plant Physiol. 43, 1185–1207
Higashi, S. (1966) Electron microscopic studies on the infection thread developing in the root hair of Trifolium repens L. infected with Rhizobium trifolii. J. Gen. Appl. Microbiol. 12, 147–156
James, E.K., Sprent, J., Sutherland, J., McInroy, S., Minchin, F. (1992a) The structure of nitrogen fixing root nodules on the aquatic mimosoid legume Neptunia plena. Ann. Bot. 69, 173–180
James, E.K., Minchin, F. Sprent, J. (1992b) The physiology and nitrogen-fixing capability of aquatically and terrestrially-grown Neptunia plena: the importance of nodule oxygen supply. Ann. Bot. 69, 181–187
James, E., Shaw, J., Catellan, A., De Faria, S., Sprent, J.I. (1993) The infection of aquatic and terrestrial Neptunia species by Rhizobium. In: New horizons in nitrogen fixation, p. 351, Palacios, R., Mora, J., Newton, W., eds. Kluwer, Dordrecht
Loureiro, M.D., De Faria, S., James, E.K., Pott, A., Franco, A.A. (1994) Nitrogen-fixing stem nodules of the legume, Discolobium pulchellum Benth. New Phytol. 128, 283–295
McVaugh, R. (1987) Flora Novo-Galiciana. In: Leguminosae, vol. 5, pp. 225–227, University of Michigan Press, Ann Arbor
Minchin, F., Witty, J., Sheehy, J., Muller, M. (1983) A major error in the acetylene reduction assay: decreases in nodular nitrogenase activity under assay conditions. J. Exp. Bot. 13, 501–512
Naisbitt, T., Sprent, J.I. (1993) The long term effects of nitrate on the growth and nodule structure of the caesalpinioid herbaceous legume Chamaecrista fasciculata Michaux. J. Exp. Bot. 44, 829–836
Napoli, C., Dazzo, F., Hubbell, D. (1975a) Ultrastructure of infection and common antigen relationships in Aeschynomene. In: Proceedings of the 5th Australian legume nodulation conference, pp. 35–37, Vincent, J., ed., Brisbane, Australia
Napoli, C., Dazzo, F., Hubbell, D. (1975b) Production of cellulose microfibrils by Rhizobium. Appl. Microbiol. 30, 123–131
Napoli, C., Hubbell, D. (1975) Ultrastructure of Rhizobium-induced infection threads in clover root hairs. Appl. Microbiol. 30, 1003–1009
Ndoye, L., DeBilly F., Vasse J., Dreyfus B., Truchet G. (1994) Root nodulation of Sesbania rostrata. J. Bacteriol. 176, 1060–1068
Newcomb, W. (1981) Nodule morphogenesis and differentiation. In: Biology of Rhizobiaceae, pp. 247–298, Giles, K., Atherly, A., eds. Academic Press, New York
Orgambide, G., Li, J., Hollingsworth, R., Philip-Hollingsworth, S., Dazzo, F. (1994) Membrane accumulation, structures, and biological activities of chitolipooligosaccharides from wild type Rhizobium leguminosarum bv. trifolii ANU843. In: Proceedings of the 7th international symposium on molecular plant-microbe interactions, p. 27. University of Edinburgh, Scotland
Parsons, R., Sprent, J.I., Raven, J.A. (1993) Humidity and light affect the growth, development, and nitrogenase activity of stem nodules of Sesbania rostrata (Brem). New Phytol. 125, 749–755
Schaede, R. (1940) Die Knollchen der adventiven Wasserwurzein, von Neptunia Oleracea und ihre Bakterien-Symbiose. Planta 31, 1–21
Shaw, J. (1993) Factors affecting nod gene induction, particularly in rhizobia from tropical trees. Ph.D. thesis, University of Dundee, UK
Sprent, J., Raven, J. (1992) Evolution of nitrogen-fixing symbioses. In: Biological nitrogen fixation, pp. 461–496, Stacey, G., Burris, R., Evans H., eds. Chapman and Hall, New York
Streeter, J., Salminen, S.O. (1993) Effect of polysaccharide deposition by Bradyrhizobium japonicum bacteroids in soybean nodules on nodule function. Plant Physiol. Biochem. 31, 73–79
Tsien, H., Dreyfus, B., Schmidt, E. (1983) Initial stages in the morphogenesis of nitrogen fixing stem nodules of Sesbania rostrata. J. Bacteriol. 156, 888–897
Truchet, G., Camut, S., DeBilly, F., Odorico, R., Vasse, J. (1989) The Rhizobium-legume symbiosis: two methods to discriminate between nodules and other root-derived structures. Protoplasma 149, 82–88
Turgeon, B., Bauer, W.D. (1985) Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum. Planta 163, 328–349
Vincent, J. (1970) A manual for the practical study of the root nodule bacteria. IBP Handbook No. 15, Blackwell Scientific Publications, Oxford, UK
Windler, D.R. (1966) A revision of the genus Neptunia (Leguminosae). Aust. J. Bot. 14, 339–340
Author information
Authors and Affiliations
Corresponding author
Additional information
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.
Rights and permissions
About this article
Cite this article
Subba-Rao, N.S., Mateos, P.F., Baker, D. et al. The unique root-nodule symbiosis between Rhizobium and the aquatic legume, Neptunia natans (L. f.) Druce. Planta 196, 311–320 (1995). https://doi.org/10.1007/BF00201390
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00201390