Molecular Soil Biology 2024, Vol.15, No.2, 46-58 http://bioscipublisher.com/index.php/msb 53 guianensis, further supports the diversity and functionality of nitrogen-fixing pathways in the rhizosphere (Villadas et al., 2007). 7.2 Genetic and proteomic analyses Genetic and proteomic analyses are essential for understanding the molecular mechanisms underlying nitrogen fixation. Techniques such as 16S rRNA gene sequencing and polymerase chain reaction (PCR) amplification of nifHgenes have been employed to identify and characterize nitrogen-fixing bacteria. For example, in sugarcane rhizosphere, Pseudomonas spp. were identified and their nitrogenase activity was confirmed through nifH gene expression studies (Li et al., 2017). Similarly, the genetic diversity of nitrogen-fixing bacteria in the rhizosphere of wheat plants was analyzed using denaturing gradient gel electrophoresis (DGGE) and quantitative PCR, revealing significant differences in bacterial communities between the rhizosphere and root endosphere (Rilling et al., 2018). Proteomic analyses, such as Biolog phenotyping, have also been used to assess the functional diversity of nitrogen-fixing bacteria. This approach has confirmed the metabolic versatility of strains like Bacillus megaterium and Bacillus mycoides, which can utilize diverse carbon and nitrogen sources and tolerate various environmental stresses (Singh et al., 2020). These genetic and proteomic insights are crucial for selecting and optimizing bacterial strains for effective nitrogen fixation in the pine rhizosphere. 7.3 Interaction between pine roots and nitrogen-fixing bacteria The interaction between pine roots and nitrogen-fixing bacteria is a complex and dynamic process that significantly influences plant growth and nutrient acquisition. Studies have shown that inoculation with nitrogen-fixing bacteria can enhance plant growth and nitrogen content. For instance, maize plants inoculated with Pseudomonas stutzeri A1501 exhibited improved growth and nitrogen accumulation, highlighting the potential benefits of such interactions in other plant systems, including pine (Ke et al., 2019). The colonization of plant roots by nitrogen-fixing bacteria involves various mechanisms, including the production of plant growth-promoting substances and the modulation of root exudates. In sugarcane, Bacillus megaterium and Bacillus mycoides were found to colonize the rhizosphere and root tissues effectively, leading to enhanced expression of genes involved in stress tolerance and pathogen resistance (Singh et al., 2020). Similarly, the co-occurrence of nitrogen-fixing and 1-aminocyclopropane-1-carboxylate deaminating bacteria in the maize rhizosphere suggests a synergistic interaction that promotes plant health and growth (Renoud et al., 2020). In the context of pine, understanding these interactions can provide valuable insights into developing biofertilizers and sustainable agricultural practices. The compartmentalization of bacterial communities between the rhizosphere and root endosphere, as observed in wheat plants, indicates that specific bacterial strains may be more effective in certain root zones, thereby optimizing nitrogen fixation and plant growth (Rilling et al., 2018). 8 Case Study: Successful Strain Application 8.1 Detailed case study of a successfully verified nitrogen-fixing strain One of the most successful applications of nitrogen-fixing bacteria in the rhizosphere of pine trees involves the strain Pseudomonas stutzeri A1501. This strain has been extensively studied for its ability to promote plant growth and enhance nitrogen acquisition in various crops, including maize. The strain was isolated and characterized for its nitrogen-fixing capabilities, which were confirmed through the presence and activity of the nifH gene, a key marker for nitrogen fixation (Ke et al., 2019). The inoculation of maize with P. stutzeri A1501 resulted in significant improvements in plant growth and nitrogen content, demonstrating the strain's potential for application in other plant species, including pine trees. 8.2 Steps from isolation to field application The process of isolating and applying Pseudomonas stutzeri A1501 to pine trees involves several critical steps. Initially, the strain was isolated from the rhizosphere of maize plants using selective media that favor the growth of nitrogen-fixing bacteria. The isolated strain was then subjected to genetic and phenotypic characterization to
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