DNA hybridization with the cloned nodulation region of Rhizobium melioti as a probe revealed DNA homology with four Hindm fragments, 12.5, 6.8, 5.2, and 0.3 kilobases (kb) in size, of the symbiotic plasmid pRjaUSDA193. Both hybridization and complementation studies suggest that the common nodulation genes nodABC and nodD of R. fredi USDA 193 are present on the 5.2-kb HindIII and 2.8-kb EcoRI fragments, respectively, of the Sym plasmid. Both fragments together could confer nodulation ability on soybeans when present in Sym plasmid-cured (Sym-) and wild-type (Sym+) Rhizobium strains or in a Ti plasmid-cured Agrobacterium tumefaciens strain. Furthermore, the 2.8-kb EcoRI fragment alone was able to form nodulelike structures on Glycine mar L. cv. "Peking" (soybean). Microscopic examination of these nodules revealed bacterial invasion of the cells, probably via root hair penetration. Bacterial strains harboring plasmids carrying the 5.2-and 2.8-kb nod fragments elicited root-hair-curling responses on infection. These data suggest that the genes responsible for host range determination and some of the early events of nodulation may be coded for by the 5.2-kb Hindm and 2.8-kb EcoRI fragments.Bacteria of the genus Rhizobium form symbiotic associations with leguminous plants and fix nitrogen in specialized structures called root nodules. This is a complex, multistage process involving induction of root hair deformation (curling, branching, or both), infection of root hairs, development and differentiation of root nodules, proliferation of bacteria within the host cells, and finally, the reduction of molecular nitrogen into ammonia (3, 37, 38). Recently, a new group of fast-growing rhizobia that nodulate soybeans was isolated in mainland China (20). These fast-growing soybean strains, previously known as Rhizobium japonicum, have been redesignated Rhizobiumfredii (35). Studies of the nodulation process in fast-growing Rhizobium strains have demonstrated the presence of a series of genetic loci coding for nodulation (nod) and nitrogenase (nif) genes, on a very large symbiotic (pSym) plasmid (2,7,22,23,30,33). The nod and nif genes of R. fredii strains have also been shown to be present on a large plasmid (26,28). Strain USDA 194 is an exception, in which the symbiotic genes are present either on a megaplasmid or on the chromosome (25). R. fredii USDA 193, used in this study, nodulates a highly inbred soybean (Glycine max L. cv. "Peking") effectively and the North American cultivars of soybean ineffectively (20).Long et al. (23) cloned a region of the symbiotic plasmid of Rhizobium meliloti 1021(pRmSL26), which complements two nodulation-deficient mutants of R. meliloti. Transposon mutagenesis and DNA sequence analysis (8, 19) revealed a cluster of four nod genes, nodABC and nodD, located on this fragment, with nodD read divergently from nodABC. Others have found that mutations in nodA, -B, and -C genes fail to induce root-hair-curling (Hac-) as well as nodule formation

DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the 'core' T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the 'core' T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.

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