Molecular Soil Biology 2025, Vol.16, No.4, 188-198 http://bioscipublisher.com/index.php/msb 1 94 stored more nitrogen in plant tissues. As a result, soybean biomass, yield, and seed protein content increased. PCR and sequencing showed that the inserted genes were stable and expressed correctly. The strains kept high performance under different conditions, and high nitrate or drought had little effect on activity—problems that often reduce the efficiency of normal strains (Alam et al., 2015; Igiehon et al., 2019; Shome et al., 2022). Compared with strains improved through selection or mutagenesis, the engineered strains had stronger nitrogen fixation, better nodulation, and higher stress resistance. Earlier work with mutagenized Rhizobium japonicum increased nitrogenase activity but often lost stability under stress (Maier and Brill, 1978). Here, targeting multiple genes improved both enzyme function and field competitiveness (Igiehon et al., 2019; Shome et al., 2022). Adding hydrogenase systems, which can improve nitrogen fixation and crop yield (Albrecht et al., 1979), gave results similar to past studies. Although some commercial and high-performing local strains can raise yield, their results vary with soil, variety, and climate (Thuita et al., 2011; Alam et al., 2015). In contrast, the engineered strains in this study performed well across sites and seasons. Co-inoculation with plant growth–promoting and phosphate-solubilizing bacteria further supported nutrient uptake and growth (Shome et al., 2022; Qin et al., 2023; Zhang et al., 2023). The introduction of exogenous genes may affect soil microbial diversity and ecological balance, and even have the potential risk of gene horizontal transfer (Damanhuri et al., 2020). In this study, no significant negative effects of engineered strains on the structure and function of soil microbial community were found in the multi-point field experiment, and they can cooperate with the local microbial community without competitive exclusion (Alam et al., 2015; Qin et al., 2023). However, long-term ecological monitoring and biosafety under large-scale application still need to be paid continuous attention. Although the environmental adaptability and competitiveness of engineered strains have been improved, their colonization ability and ecological impact in different ecological areas may be different. In order to ensure ecological security, scientific strain management and monitoring measures should be formulated to prevent the irreversible impact of exogenous strains on the local ecosystem. In the future, it is necessary to strengthen the systematic assessment of gene flow, ecological adaptability and long-term environmental impact to ensure the sustainable application of engineered rhizobia (Damanhuri et al., 2020; Abd-Alla et al., 2023). Although this study has made positive progress in improving the nitrogen fixation efficiency of soybean by engineered Rhizobium strains, there are still some limitations. The experiments mainly focused on greenhouse and limited field conditions, and have not covered a wider ecological area and diversified soil types. Climate, soil and crop management methods in different regions may affect the colonization and nitrogen fixation effect of engineered strains, and large-scale field validation with multiple sites and seasons is needed (Alam et al., 2015; Qin et al., 2023). The long-term genetic stability and ecological security of molecular transformation need to be continuously tracked. Although no obvious ecological risk was found in the short term, the long-term expression and potential gene flow of foreign genes in the natural environment still need to be vigilant. The interaction between engineered strains and soybean varieties and the synergy mechanism with local microbial communities also need to be further analyzed. The engineered Rhizobium strains showed great potential in improving nitrogen fixation efficiency of soybean, promoting crop growth and reducing fertilizer dependence. The results of field trials showed that the engineered strains could steadily improve the yield and quality of soybean in a variety of environments, providing a new path for achieving green high yield and sustainable agricultural development (Thuita et al., 2011; Alam et al., 2015; Kolapo et al., 2025). Combined with improved soybean varieties and precision fertilization management, engineered rhizobia is expected to become an important biological input for soybean production in the future, and help green transformation of agriculture and food security (Abd-Alla et al., 2023; Kolapo et al., 2025). The large-scale application still needs to solve the technical problems of strain production, storage, transportation and field inoculation, and strengthen the ecological security management and policy support. It is suggested that in the future, multidisciplinary cooperation should be carried out in strain breeding, inoculation technology optimization, ecological risk assessment and industrialization promotion, so as to promote the wide application of engineered
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