Molecular Soil Biology 2024, Vol.15, No.2, 46-58 http://bioscipublisher.com/index.php/msb 48 In some studies, seedlings of pine and other tree species are planted in controlled environments, such as pots containing different soil types, to observe the influence of the rhizosphere on microbial communities. After a growing season, the rhizosphere soil is collected for analysis (Priha et al., 1999). This method allows for a controlled comparison of microbial communities across different soil types and plant species. 3.2 Isolation techniques for nitrogen-fixing bacteria The isolation of nitrogen-fixing bacteria from the pine rhizosphere involves both cultivation-dependent and cultivation-independent methods. Cultivation-dependent methods include the use of specialized culture media that promote the growth of nitrogen-fixing bacteria. For instance, trap experiments with legumes can be employed to isolate rhizobia, a group of nitrogen-fixing bacteria. These experiments involve growing legumes in the presence of rhizosphere soil to encourage the formation of root nodules, which are then analyzed for the presence of nitrogen-fixing bacteria (Priha et al., 1999). Additionally, high-throughput sequencing techniques, such as 16S rRNA gene sequencing, are used to identify and quantify bacterial communities in the rhizosphere. This method provides a comprehensive profile of the microbial community, including both culturable and non-culturable bacteria. Functional annotation tools, such as FAPROTAX, can further classify these bacteria based on their metabolic functions, including nitrogen fixation (Figure 1) (Liu et al., 2022). 3.3 Criteria for preliminary selection of nitrogen-fixing strains The preliminary selection of nitrogen-fixing strains from the pine rhizosphere is based on several criteria. The ability to fix nitrogen is a primary criterion. This can be assessed through the presence of nitrogenase activity, which is the enzyme responsible for converting atmospheric nitrogen into a form usable by plants. Bacteria exhibiting high nitrogenase activity are considered strong candidates for further study (Kaplan et al., 2013). The potential for plant growth promotion is evaluated. This includes the ability to solubilize phosphate, produce siderophores (which chelate iron), and secrete enzymes such as cellulase. These traits are beneficial for plant health and growth, making the bacteria more valuable as biofertilizers (Kaplan et al., 2013; Liu et al., 2022). The adaptability and resilience of the bacteria in different soil types and environmental conditions are considered. Bacteria that can thrive in both organic and mineral soils, and under varying environmental stresses, are preferred. This ensures that the selected strains can be effectively used in diverse agricultural settings (Priha et al., 1999). 4 Identification and Characterization of Isolated Strains 4.1 Molecular identification methods (e.g., 16S rRNA sequencing) Molecular identification of nitrogen-fixing bacteria isolated from the pine rhizosphere primarily involves 16S rRNA gene sequencing. This method is widely recognized for its accuracy in identifying bacterial species and understanding their phylogenetic relationships. For instance, in a study on Pseudomonas spp. isolated from the sugarcane rhizosphere, 16S rRNA gene sequencing was employed to identify the strains and analyze their phylogenetic diversity. The results confirmed that all strains were similar to Pseudomonas spp. (Li et al., 2017). Similarly, strains E20 and E50 (T) isolated from the rhizosphere of salt meadow plants were identified as members of the genus Cellvibrio through 16S rRNA gene sequence analysis, showing high similarity with Cellvibrio gandavensis (Suarez et al., 2014). In another study, nitrogen-fixing bacteria from the maize rhizosphere were identified as members of the genus Paenibacillus using specific primers based on the 16S rRNA gene (Weid et al., 2002). This method was also used to identify nitrogen-fixing bacteria from the rhizosphere of various agricultural crops in Korea, confirming the presence of Stenotrophomonas maltophilia, Bacillus fusiformis, and Pseudomonas fluorescens (Park et al., 2005). Additionally, 16S rRNA gene sequencing was utilized to characterize nitrogen-fixing bacteria from banana and pineapple rhizospheres, identifying species such as Herbaspirillum seropedicae and Burkholderia brasilensis (Cruz et al., 2002).
RkJQdWJsaXNoZXIy MjQ4ODYzMg==