Bradyrhizobium sp. DOA9 has a broad host range and a highly divergent nodulation (nod) gene on the megaplasmid (pDOA9). Duplicated copies of the general regulator nodD (nodD1 and nodD2) and nodA (nodA1 and nodA2) genes were found on pDOA9. The nodA gene is encoding an acyltransferase that controls the transfer of an acyl chain to the chitooligosaccharides (COs) structure and contribute to the determination of host specify. In general, compatible recognition between transcriptional regulators NodD and plant flavonoid inducer able to activate the function of other nodulation (nod) genes which responsible to produce lipo-chitooligosaccharides (LCOs), calls Nod-Factor, a major determinants of host range, infection, and nodulation. In this study, the function of the duplicated nodD (nodD1 and nodD2) and nodA (nodA1 and nodA2) genes were disrupted to determine their role on the symbiotic nodulation. The results showed that the mutation of nodD1 or nodD2 did not affected to the symbiotic nodulation, indicating the functional redundancy of the regulatory nodD1 and nodD2 genes in strain DOA9. Whereas nodA2 and nodB genes plays as a central role in the Nod-Factor biosynthesis and the symbiotic nodulation in all plant tests. Moreover, Nod-Factor produced from DOA9 under genistein induction shared a high similarity with the major Nod-Factor of Rhizobium sp. NGR234. Interestingly, mutation of nodA1 (?nodA1) did not affect to the nodulation but lead to induce the production of Nod-Factor belonged to the wild type, with the absence of Nod-Factor bearing the C18:1 acyl group. All these results suggest that the host range of the strain DOA9 might acquire from the functional redundancy of regulatory nodD genes and the Nod-Factor structure. © 2022 Elsevier B.V.