Molecular Soil Biology 2024, Vol.15, No.2, 46-58 http://bioscipublisher.com/index.php/msb 47 2 Literature Review 2.1 Overview of nitrogen fixation processes Biological nitrogen fixation (BNF) is a critical process in which atmospheric nitrogen (N2) is converted into ammonia (NH3) by nitrogenase enzymes, making nitrogen available to plants in a form they can absorb and utilize. This process is primarily mediated by diazotrophs, a diverse group of microorganisms capable of fixing nitrogen either symbiotically or freely in the soil. Symbiotic nitrogen fixation (SNF) involves the formation of specialized structures called nodules on plant roots, where bacteria such as rhizobia and Frankia reside and perform nitrogen fixation (Franche et al., 2009; Lindström and Mousavi, 2009; Aasfar et al., 2021). Free-living nitrogen-fixing bacteria, such as Azotobacter species, also play a significant role in enhancing soil fertility and plant nutrition by fixing nitrogen independently of plant hosts (Aasfar et al., 2021). The regulation of BNF is complex and involves sophisticated genetic and environmental controls to ensure efficiency and adaptability to varying conditions (Dixon and Kahn, 2004; Kahindi, 2020). 2.2 Previous studies on rhizosphere nitrogen-fixing bacteria in various plant species Research on rhizosphere nitrogen-fixing bacteria has been extensive, covering a wide range of plant species. In leguminous plants, rhizobia form symbiotic relationships, leading to the development of root nodules where nitrogen fixation occurs (Lindström and Mousavi, 2009; Aasfar et al., 2021). Studies have shown that these symbiotic interactions significantly enhance soil nitrogen levels and improve plant growth and yield. For non-leguminous plants, nitrogen-fixing bacteria such as Pseudomonas stutzeri and Bacillus species have been identified as beneficial for plant growth and nitrogen acquisition. For instance, inoculation with Pseudomonas stutzeri A1501 has been shown to improve maize growth and nitrogen content, demonstrating the potential of these bacteria as biofertilizers (Ke et al., 2019). Similarly, Bacillus megateriumand Bacillus mycoides have been found to enhance nitrogen fixation and provide biocontrol properties in sugarcane, highlighting their dual role in promoting plant health and growth (Singh et al., 2020). The diversity and functional capabilities of nitrogen-fixing bacteria in the rhizosphere of various crops, including maize and sugarcane, underscore their importance in sustainable agriculture (Kuan et al., 2016; Renoud et al., 2020). 2.3 Specific challenges and knowledge gaps in pine rhizosphere studies Despite the extensive research on nitrogen-fixing bacteria in the rhizospheres of various crops, studies focusing on pine trees remain limited. One of the primary challenges in pine rhizosphere studies is the identification and isolation of effective nitrogen-fixing bacteria that can thrive in the unique soil conditions associated with pine forests. Pines often grow in nutrient-poor, acidic soils, which can limit the diversity and activity of nitrogen-fixing bacteria (Suarez et al., 2014). Additionally, the symbiotic relationships between pine roots and nitrogen-fixing bacteria are not as well understood as those in leguminous plants, posing a significant knowledge gap. The potential for co-selection of beneficial microbial traits, such as nitrogen fixation and stress tolerance, in the pine rhizosphere also remains underexplored (Renoud eta al., 2020). Addressing these challenges requires a comprehensive understanding of the microbial communities in pine rhizospheres and their interactions with host plants. Future research should focus on isolating and characterizing nitrogen-fixing bacteria from pine rhizospheres, understanding their ecological roles, and developing effective inoculants to enhance pine growth and soil fertility (Dixon and Kahn, 2004; Ke et al., 2019; Aasfar et al., 2021). 3 Screening of Nitrogen-Fixing Bacteria 3.1 Description of pine rhizosphere sampling methods The sampling of the pine rhizosphere involves several meticulous steps to ensure the accurate collection of microbial communities associated with the root systems. Typically, the process begins with the selection of healthy pine trees, ensuring that the samples are representative of the natural environment. The soil surrounding the roots, known as the rhizosphere, is carefully excavated to a depth of approximately 1~3 mm from the root surface. This region is rich in microbial activity and is crucial for studying plant-microbe interactions (Kaplan et al., 2013).
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