Abstract
Dual occupancy of Parasponia andersonii nodules with different Bradyrhizobium strains and Rhizobium leguminosarum biovar trifolii was frequently obtained when two strains were inoculated into plants grown aseptically in tubes. Since reisolates of Bradyrhizobium strains from dually occupied nodules acquired the ability to nodulate Trifolium repens, the spatial relationship of the two species of bacteria during nodule initiation and development was investigated and their proximity was demonstrated. By using light microscopy and electron microscopy and immunogold labeling, R. leguminosarum biovar trifolii NGR66 inoculated alone onto P. andersonii produced small ineffective nodules, with bacteria embedded in matrix material in intercellular spaces and in a few nonliving host cells rather than in infection threads (CP299). In dual infections, the two bacterial species were shown to be adjacent to one another in the matrix of nodule intercellular spaces and in some host nodule cells. However, when two different Bradyrhizobium strains occupied a single nodule, they were located in different lobes of the same nodule. Immunogold labeling showed that Parasponia hemoglobin was localized in the cytoplasm of young infected nodule cells. This suggests that the nitrogen-fixing phase of Parasponia nodule cells is short-lived and correlates with previous acetylene reduction data from nodule slices. Hemoglobin was associated only with areas of nodule tissue infected with the effective nitrogen-fixing strain CP299 and absent from areas infected with R. leguminosarum biovar trifolii.