EFFECT OF NIF GENE STRUCTURE VARIATION ON SYMBIOTIC EFFECTIVENESS IN RHIZOBIUM FREDII AND BRADYRHIZOBIUM JAPONICUM

The purpose of this study was to evaluate the effect of nif gene structure variation on symbiotic effectiveness in Rhizobium fredii and to determine if nif gene structure variation is responsible for differences in symbiotic effectiveness in Bradyrhizobium japonicum strain USDA 110 colonial derivatives.; In R. fredii, a mutant of strain USDA 206, cured of its 197 MDa "sym" plasmid (USDA 206C) which had retained its symbiotic effectiveness was compared to wild type USDA 206. Statistically higher values were obtained for USDA 206 for nodule number, nodule mass, nitrogenase activity per plant, nitrogenase specific activity and plant dry weight. An apparent correlation between loss of "sym" plasmid-borne nif gene copies and reduction of symbiotic effectiveness was found. Delayed nodulation by strain USDA 206C relative to strain USDA 206 also indicated an association with the loss of plasmid-borne nodulation functions and the reduced effectiveness of USDA 206C.; In B. japonicum strain USDA 110 four colony morphology variants have previously been detected. I-110 and S-110 variants have previously been found to be five to tenfold more efficient in nitrogen fixation than L1-110 and L2-110 variants. EcoRI, BamHI, SmaI, HindIII and XhoI restriction enzyme digests of total DNA revealed similar patterns for all four morphology variants. Hybridization of a nif DH probe from R. meliloti revealed the same fragment patterns for each variant in BamHI and EcoRI digests. These patterns corresponded to those previously published for USDA 110. HindIII digests showed an extra 6-kb fragment in L1-110 and L2-110. This band was not detected in I-110 or S-110 digests. However, this band was very faint when compared to the 8.5-kb fragment in HindIII digests corresponding to nif D homology. Subsequent hybridization to HindIII digests with purified nif D probe from R. meliloti have shown the presence of four minor bands of homology in L1-110 (6.0, 6.5, 7.0 and 7.5 kb) not present in S-110. Both L1-110 and S-110 possess a major 8.5-kb band of nif D homology in HindIII digests. Hybridization of a purified nif D probe to HindIII digests revealed a single major band of homology for both S-110 and L1-110 at 0.75 kb. The minor bands found in L1-110 indicate duplications of nif D sequences not found in S-110. Additional plant data were also collected and substrain L1-110 was shown to be completely inefficient in nitrogen fixation, whereas strain I-110 was found to be symbiotically competent.