Molecular Soil Biology 2025, Vol.16, No.4, 188-198 http://bioscipublisher.com/index.php/msb 1 89 The capacity of native rhizobia depends on genetics, stress factors such as drought or high nitrate, and compatibility with soybean cultivars. Some strains perform poorly in high-nitrate soils or under harsh conditions (Maier and Brill, 1978; Nguyen et al., 2019; Igiehon et al., 2019). Molecular tools can improve these traits by enhancing stress tolerance, increasing nitrogenase activity, or strengthening signaling with the host. Adding an efficient hydrogenase system reduces hydrogen loss during fixation (Albrecht et al., 1979). Adjusting signaling pathways can improve root interaction (Wang et al., 2021; Zhang et al., 2023). Co-inoculation with plant growth–promoting or phosphate-solubilizing bacteria further improves nutrient uptake and stress tolerance (Igiehon et al., 2019; Shome et al., 2022; Zhang et al., 2023). This study examines recent developments in engineering rhizobial strains to improve nitrogen fixation in soybean. It discusses the basic nitrogen fixation process, soybean nitrogen requirements, mechanisms of rhizobium–soybean interaction, genetic improvement methods, and the molecular basis of these strategies. Both laboratory and field research are considered to evaluate the potential and challenges of using engineered strains in sustainable farming systems. 2 Literature Review 2.1 Rhizobium species symbiotic with soybean The main rhizobia used for soybean are Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens. Both can form root nodules and fix nitrogen, but different strains vary in nodulation efficiency, nitrogen fixation capacity, and tolerance to stress. For instance, B. japonicum USDA110 is widely used as a commercial inoculant but tends to show reduced activity in soils with high nitrate content (Nguyen et al., 2019). Recently, some new Bradyrhizobium strains have been identified that can keep high activity under such conditions, giving farmers more options to match local soil conditions and fertilizer use. Other species, such as Rhizobium sp. and Ensifer meliloti, can also form effective partnerships with soybean. In some cases, they perform better under specific environmental stresses (Igiehon et al., 2019). Many of these strains also have plant growth–promoting traits, including phosphate solubilization, stress resistance, and production of plant hormones. These traits can help soybeans grow better in poor soils. 2.2 Mechanism of nitrogen fixation Soybean roots release flavonoids that trigger the nod genes in rhizobia. These genes control the production of Nod factors, which signal the plant to start forming nodules (Wang et al., 2021; Zhang et al., 2023). Signals from the shoot, including GmSTF3/4 and GmFTs, help adjust nodulation so that nitrogen fixation matches the plant’s available carbon supply (Wang et al., 2021). Inside the nodules, the nitrogenase enzyme complex—made of Fe protein and MoFe protein—reduces nitrogen gas to ammonia under low-oxygen conditions. Some strains have a hydrogenase system that recycles hydrogen, making nitrogen fixation more energy efficient (Albrecht et al., 1979; Neves et al., 1985). Plant–rhizobium communication continues during the entire process, involving hormones such as indole-3-acetic acid (IAA) and salicylic acid (SA), as well as secondary metabolites and coordinated carbon–nitrogen metabolism (Zhang et al., 2023). 2.3 Genetic engineering strategies Genetic improvement focuses on making rhizobia form nodules faster, increase nitrogenase activity, and tolerate stress. Mutagenesis has been used to create B. japonicum mutants that nodulate earlier and keep fixing nitrogen even when external nitrogen is present (Maier and Brill, 1978). Adding hydrogenase systems to engineered strains has been reported to improve nitrogen fixation efficiency and increase crop yield (Albrecht et al., 1979). Gene editing and transgenic methods can change important genes like nod and nif, or add stress-resistance genes such as exoX and htrA, so rhizobia can keep working under high temperature, drought, and other challenging conditions (Igiehon et al., 2019). Another approach is to inoculate soybeans with rhizobia together with beneficial microbes, such as phosphate-solubilizing bacteria, which help plants take up more nutrients and cope with stress more effectively (Shome et al., 2022; Zhang et al., 2023).
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