Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or Rhizobium. The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.Neptunia natans (L.f.) Druce is an aquatic legume native to several continents of the humid tropics and is used for both human consumption and as green manure for rice cultivation in Asiatic countries. This legume is unusual in that it normally develops buoyant floating stems that grow profusely on the surface of freshwater ponds, and in this aquatic environment it develops many stem-associated nitrogen-fixing nodules.The developmental morphology of the infection process leading to formation of nitrogen-fixing nodules in N. natans has been examined under strict gnotobiotically controlled conditions, and several unique aspects of this specialized, aquatic plant-bacterium symbiosis distinguish it from other legume root-nodule symbioses (22). After colonizing the root and floating stem surfaces, the aquatic bacterium symbiont enters the primary root cortex and stem interior through natural wounds caused by splitting of the epidermis and emergence of young lateral and adventitious roots, respectively, and then stimulates early development of nodules in the cortex at the base of these roots primordia, but not in the stem itself (22). Following crack entry through the nodule periphery, the bacteria penetrate internal nodule host cells; induce formation of bona fide tubular infection threads that disseminate them further intracellularly; and then release the bacteria into infection droplets, where they multiply. The endosymbiotic bacteria transform into nitrogen-fixing bacteroids within symbiosomes that eventually become filled with an unusual fibrillar matrix towards the end of their active nitrogen-fixing cycle in the aquatic e...

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Abstract.We examined the development of the aquatic N2-fixing symbiosis between Rhizobium sp. (Neptunia) 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

Phosphate solubilizing ability of different isolates of fungi associated with legume root nodules was studied in vitro. Among the fungi tested, isolates of Penicillium lilacinum, Aspergillus sp., A. flavus, A. niger, A. terreus, and A. nidulans solubilized insoluble tricalcium phosphate. When soluble potassium dihydrogen phosphate was present with tricalcium phosphate in the medium, some of the fungi failed to solubilize phosphate.

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