Molecular Soil Biology 2025, Vol.16, No.4, 188-198 http://bioscipublisher.com/index.php/msb 1 90 2.4 Challenges in field use and safety Although engineered rhizobia have shown excellent nitrogen fixation ability in laboratory and greenhouse conditions, their application in the field still faces multiple challenges. The genetic stability and environmental adaptability of the strain are the key factors affecting its field performance. Some efficient strains are not competitive enough in complex soil microbial communities, and are difficult to colonize for a long time or be replaced by local flora, resulting in unstable nitrogen fixation effect (Thuita et al., 2011; Nguyen et al., 2019). Environmental factors such as soil pH, temperature, moisture, nutrients, etc. will affect the survival and symbiotic efficiency of rhizobia (Igiehon et al., 2019; Hu et al., 2023). Ecological security is also an important issue for the application of engineered rhizobia. Exogenous or genetically modified strains may have potential effects on soil microbial diversity and ecosystem functions, and even cause biological safety hazards such as horizontal gene transfer (Abd-Alla et al., 2023). Improving the environmental adaptability and competitiveness of strains, optimizing inoculation technology, and collaborative breeding with local varieties are also the key directions to achieve efficient field application of engineered rhizobia (Thuita et al., 2011; Nguyen et al., 2019; Abd-Alla et al., 2023). 3 Materials and Methods 3.1 Strain selection and genetic modification 3.1.1 Selection criteria The starting strain should have high nitrogen-fixing ability, form many nodules, match well with the soybean variety, and tolerate heat, drought, or acidic soils. Previous field and greenhouse work identified Bradyrhizobium japonicum USDA110 and its mutants, Rhizobium sp. R1, and R. cellulosilyticum R3 as good candidates. These strains perform well even when nitrate is high or during drought (Maier and Brill, 1978; Igiehon et al., 2019; Nguyen et al., 2019). Competitiveness in soil and strong root colonization are also important traits (Alam et al., 2015; Nguyen et al., 2019). Strains that have complete nif, nod, stress-resistance genes, and plant growth-promoting genes are preferred (Igiehon et al., 2019). 3.1.2 Molecular tools Common molecular techniques for rhizobium engineering include CRISPR/Cas, homologous recombination, and plasmid transfer. CRISPR/Cas can make precise changes to the genome (Igiehon et al., 2019). Homologous recombination is used to insert or replace specific genes, while plasmid transfer can bring in large gene clusters. In some cases, these methods are combined-for example, removing negative regulatory genes with CRISPR/Cas9 and introducing hydrogenase genes using plasmids (Albrecht et al., 1979). 3.1.3 Target genes The core of rhizobia engineering is the precise modification of key functional genes. Nitrogenase gene (nif gene) is the core of nitrogen fixation reaction. Enhancing its expression or activity can directly improve nitrogen fixation efficiency (Maier and Brill, 1978; Igiehon et al., 2019). Regulatory genes (such as nod gene and signal transduction related genes) determine the nodulation ability and symbiotic adaptability of the strain and soybean. The nodulation process and symbiotic signal exchange can be optimized by regulating these genes (Wang et al., 2021; Zhang et al., 2023). Stress resistance related genes (such as exoX, htrA, etc.) endow the strain with the ability of survival and nitrogen fixation under high temperature, drought, acid and other stresses, which is the key to improve the stability of field application (Igiehon et al., 2019). The research also focused on the introduction and expression of hydrogenase system genes, and improved energy utilization and nitrogen fixation capacity by recycling hydrogen, a by-product of nitrogen fixation (Albrecht et al., 1979). 3.2 Experimental design 3.2.1 Laboratory culture Rhizobia are grown in YEM or TY medium at 28 ℃, pH 6.8~7.2 (Igiehon et al., 2019; Hu et al., 2023). For engineered strains, antibiotics or other selection agents are added. Growth is monitored by measuring density and viability. Gene presence and expression are checked using PCR and qPCR.
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