Molecular Soil Biology 2025, Vol.16, No.4, 188-198 http://bioscipublisher.com/index.php/msb 1 93 commercial inoculants). In both conditions, engineered strains consistently produced more nodules and greater nodule fresh weight than controls. Some mutants initiated nodulation earlier, with nodules that were larger, heavier, and more uniformly distributed, indicating improved nodulation efficiency (Maier and Brill, 1978; Alam et al., 2015). Engineered strains maintained high nodulation rates across different soybean varieties and soil types. Nodule structure analysis showed larger and darker nodules, features associated with stronger nitrogen fixation. High efficiency was maintained even in high-nitrate soils and under drought stress (Igiehon et al., 2019; Nguyen et al., 2019). 4.3 Nitrogenase activity and efficiency The activity of nodule nitrogenase was determined by acetylene reduction method (ARA). The results showed that the ARA value of the engineered strain inoculation group was significantly higher than that of the control group, and the nitrogenase activity of some strains increased by 30%~100% (Maier and Brill, 1978; Albrecht et al., 1979; Alam et al., 2015). The engineered strains could maintain high nitrogenase activity with or without exogenous nitrogen source, and showed good nitrogen fixation stability. In particular, the ARA value and nitrogen accumulation of the strains with hydrogenase system were significantly higher than those without hydrogenase system, indicating that the hydrogen recovery mechanism effectively improved the nitrogen fixation efficiency (Albrecht et al., 1979). The results of field experiments showed that the engineered strains could maintain high nitrogenase activity in different ecological environments, and had good interaction with soybean varieties. Some new strains can still maintain efficient nitrogen fixation in high nitrate nitrogen environment, breaking through the bottleneck of traditional strains susceptible to nitrogen fertilizer (Nguyen et al., 2019). 4.4 Effects on soybean growth and yield In both greenhouse and field trials, engineered strains promoted stronger plant growth. Inoculated plants had higher shoot and root biomass, with dry and fresh weights increasing by 20%~60% compared to controls (Maier and Brill, 1978; Alam et al., 2015). Yield components also improved: pods per plant, seeds per pod, 100-seed weight, and total yield were all higher with engineered strains. Some treatments achieved over 30% yield gains (Alam et al., 2015; Shome et al., 2022). Seed quality improved as well. Protein content increased by up to 7% in some treatments (Shome et al., 2022). Nitrogen levels in both plants and soil were higher, confirming effective nitrogen fixation and transfer from the bacteria to the soybean host. 4.5 Environmental adaptability and survival Engineered strains showed strong adaptability in challenging environments. They maintained colonization, nodulation, and nitrogen fixation under high temperature, drought, and acidic soil conditions (Igiehon et al., 2019). Some strains grew at 45 ℃ and pH 4 while still supporting soybean seed germination. Multi-site field trials confirmed their long-term survival under different soil and climate conditions. Engineered strains coexisted with native microbial communities without causing ecological imbalance. They competed effectively, replacing less efficient local strains and improving overall nitrogen fixation (Alam et al., 2015; Nguyen et al., 2019). Ecological safety monitoring detected no harmful effects on soil microbial diversity or ecosystem functions, supporting the potential for safe, large-scale application of engineered rhizobia. 5 Discussion In this work, rhizobium strains were modified by changing genes for nitrogen fixation (nif), nodulation (nod), and stress tolerance (exoX, htrA). These engineered strains formed more nodules, had higher nitrogenase activity, and
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