Molecular Soil Biology 2024, Vol.15, No.2, 46-58 http://bioscipublisher.com/index.php/msb 52 robustness. Pine seedlings were inoculated with selected nitrogen-fixing bacterial strains, such as those identified in previous studies (Villadas et al., 2007; Phillips et al., 2011; Renoud et al., 2020). Control groups without bacterial inoculation were also established to serve as baselines for comparison. The trials spanned multiple growing seasons to capture both short-term and long-term effects of bacterial inoculation on pine growth and soil health. 6.2 Monitoring and data collection methods Monitoring and data collection were critical components of the field trials. Soil and root samples were collected at regular intervals to assess bacterial colonization and nitrogen fixation activity. Molecular techniques, such as quantitative PCR and metagenomic sequencing, were employed to quantify the presence and activity of nitrogen-fixing genes (nifH) in the rhizosphere (Phillips et al., 2011; Li et al., 2017; Renoud et al., 2020). Additionally, soil nutrient levels, particularly nitrogen content, were measured using standard soil analysis protocols. Plant growth parameters, including height, biomass, and root development, were recorded to evaluate the impact of bacterial inoculation on pine growth. Root exudation rates were also measured to understand the interaction between pine roots and the introduced acteria (Phillips et al., 2011). Environmental factors, such as soil moisture, temperature, and pH, were continuously monitored to correlate with bacterial activity and plant growth. 6.3 Results and analysis of field performance The field trials yielded significant insights into the performance of nitrogen-fixing bacteria in pine forests. Inoculated pine seedlings exhibited enhanced growth compared to the control groups, with notable increases in height and biomass. The presence of nitrogen-fixing bacteria in the rhizosphere was confirmed through molecular analyses, which showed elevated levels of nifHgene expression in inoculated plots (Villadas et al., 2007; Li et al., 2017; Renoud et al., 2020). Soil analyses revealed higher nitrogen content in the rhizosphere of inoculated pines, indicating effective nitrogen fixation by the introduced bacteria. This was further supported by increased microbial activity and enzyme production related to nitrogen cycling in the soil (Phillips et al., 2011). The enhanced root exudation observed in inoculated pines likely facilitated better microbial colonization and activity, creating a positive feedback loop that promoted plant growth and soil health (Phillips et al., 2011). Comparative analyses across different field sites highlighted the adaptability and effectiveness of the bacterial strains under varying environmental conditions. Sites with initially lower soil fertility showed the most pronounced improvements, suggesting that nitrogen-fixing bacteria could be particularly beneficial in nutrient-poor soils (Grayston and Campbell, 1996; Shi et al., 2021). The trials also demonstrated the importance of selecting appropriate bacterial strains, as different strains exhibited varying levels of nitrogen fixation and plant growth promotion (Li et al., 2017). 7 Mechanisms of Nitrogen Fixation 7.1 Detailed study of nitrogen fixation pathways in selected strains Nitrogen fixation is a critical process for converting atmospheric nitrogen (N2) into a form that plants can utilize, such as ammonia (NH3). This process is primarily facilitated by nitrogenase enzymes encoded by nif genes. In the context of pine rhizosphere, various bacterial strains have been identified with the capability to fix nitrogen. For instance, Bacillus megaterium and Bacillus mycoides have been shown to possess significant nitrogenase activity, as evidenced by the expression of the nifH gene, which is a marker for nitrogen fixation (Singh et al., 2020). Similarly, Pseudomonas stutzeri A1501 has demonstrated the ability to enhance nitrogen content in maize, indicating its potential for nitrogen fixation in other plant systems, including pine (Ke et al., 2019). The pathways of nitrogen fixation in these bacteria involve the reduction of atmospheric nitrogen to ammonia under microaerobic conditions, which is then assimilated into organic compounds. The presence of nifH gene sequences in various bacterial communities associated with different plants, such as Eperua falcata and Dicorynia
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