Molecular Soil Biology 2025, Vol.16, No.4, 188-198 http://bioscipublisher.com/index.php/msb 1 91 3.2.2 Greenhouse and field trials Greenhouse and field experiments are the key links to evaluate the nitrogen fixation efficiency and growth promoting effect of engineered Rhizobium strains. Greenhouse experiments usually use pot or nutrient solution culture system to strictly control the temperature, humidity, light and nutrient conditions, which is convenient for fine monitoring of nodulation, nitrogen fixation and plant growth (Maier and Brill, 1978; Igiehon et al., 2019; Hu et al., 2023) (Figure 1). In the field experiment, randomized block design or split block design was used to set up different inoculation treatments and control groups to investigate the colonization ability, nitrogen fixation efficiency and the impact on soybean yield of the strains in the natural environment (Nguyen et al., 2019; Kolapo et al., 2025). During the experiment, nodules, plants and soil samples should be collected regularly to analyze the number of nodules, nitrogenase activity, plant growth and yield. Multi site and multi season field trials are helpful to evaluate the adaptability and stability of strains in different ecological environments. Figure 1 The Pearson correlation coefficients for plant indexes are as follows: (a) without rhizobia inoculation at the V9 stage, (b) without rhizobia inoculation at the R5 stage, (c) with rhizobia inoculation at the V9 stage, and (d) with rhizobia inoculation at the R5 stage. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. S, sulfur concentration; SB, shoot biomass; RB, root biomass; RL, root length; SRL, specific root length; SNC, shoot N concentration; RNC, root N concentration; NNC, nodule N concentration; SBN, shoot biomass N accumulation; RBN, root biomass N accumulation; NBN, nodule biomass N accumulation; NA., nitrogenase activity; N03, number of nodules 0-3 mm in diameter; NL3, number of nodules larger than 3 mm in diameter; TNN, total number of nodules; BN03, biomass of nodules 0-3 mm in diameter; BNL3, biomass of nodules larger than 3 mm in diameter; TBN, total biomass of nodules (Adopted from Hu et al., 2023)
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