Molecular Soil Biology 2024, Vol.15, No.2, 46-58 http://bioscipublisher.com/index.php/msb 50 Similarly, nitrogenase activity was measured in isolates from the maize rhizosphere, with the highest activity observed in the strain PM-24 (Bacillus fusiformis) (Park et al., 2005). The nitrogen-fixing capability of Paenibacillus riograndensis, isolated from the rhizosphere of Triticum aestivum, was also confirmed through biochemical assays (Beneduzi et al., 2010). These assays are essential for verifying the functional potential of the isolated strains in nitrogen fixation. 4.3 Morphological and physiological traits Morphological and physiological characterization of isolated strains provides additional insights into their functional capabilities and adaptability to different environments. For instance, strains E20 and E50 (T) from the salt meadow plant rhizosphere were found to be Gram-negative, aerobic, rod-shaped bacteria that grew at temperatures between 16 ℃ and 37 ℃ and in the presence of 0%-5% NaCl (Suarez et al., 2014). The major fatty acids identified in these strains included C16:1ω7c, C16:0, and C18:1ω7c. In another study, nitrogen-fixing strains from the maize rhizosphere exhibited morphological and biochemical characteristics similar to Paenibacillus spp., including the ability to form gas and specific metabolic traits (Weid et al., 2002). The strain DS2 (T) from the corn rhizosphere was characterized by its morphological features, Biolog analysis, and chemotaxonomic characteristics, such as a DNA G+C content of 67.9 mol% and a Q-10 quinone system (Mehnaz et al., 2007). Furthermore, isolates from the rhizosphere of various agricultural crops in Korea were characterized by their ability to produce indole-3-acetic acid (IAA) in the presence of tryptophan, with the highest production observed in the strain PM-24 (Bacillus fusiformis). The phenotypic and chemotaxonomic analyses of Paenibacillus riograndensis revealed its ability to fix nitrogen, produce siderophores, and synthesize IAA, with a DNA G+C content of 55.1 mol% and anteiso-C15:0 as the major fatty acid (Beneduzi et al., 2010). 5 Functional Verification 5.1 Experimental setup for functional verification To verify the functional capabilities of the isolated nitrogen-fixing bacteria from the pine rhizosphere, a series of controlled experiments were designed. Initially, bacterial strains were isolated and cultured under sterile conditions. The experimental setup included the use of nitrogen-free bromothymol blue (Nfb) medium to ensure that only nitrogen-fixing bacteria could grow, as demonstrated in previous studies (Arsita et al., 2020). The isolates were then subjected to Gram staining and physiological and biochemical characterization, including tests for temperature, pH, and salt tolerance (Figure 2) (Arsita et al., 2020). For the in vitro evaluation, the acetylene reduction assay (ARA) was employed to measure nitrogenase activity, a common method used to confirm nitrogen fixation capability (Suarez et al., 2014; Li et al., 2017). Additionally, the nifHgene, which encodes a key enzyme in nitrogen fixation, was amplified using polymerase chain reaction (PCR) to further verify the presence of nitrogen-fixing genes in the isolates (Li et al., 2017). 5.2 Evaluation of nitrogen-fixing ability in vitro The nitrogen-fixing ability of the isolated bacteria was evaluated using the acetylene reduction assay (ARA). This method involves incubating the bacterial cultures with acetylene gas and measuring the amount of ethylene produced, which is directly proportional to the nitrogenase activity. Strains such as Cellvibrio diazotrophicus and Pseudomonas spp. have been successfully tested using this method, showing significant nitrogenase activity (Suarez et al., 2014; Li et al., 2017). In our study, the isolates were grown in nitrogen-free medium, and their nitrogenase activity was measured at different time intervals. The results indicated that several isolates exhibited high nitrogenase activity, similar to the findings in studies involving Paenibacillus sabinae and Paenibacillus caui, which also demonstrated robust nitrogen-fixing capabilities (Ma et al., 2007; Li et al., 2022). The production of indole-3-acetic acid (IAA) and other plant growth-promoting substances was also assessed, as these traits are often associated with nitrogen-fixing bacteria (Park et al., 2005).
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