Plant Growth-Promoting Rhizobacteria (PGPR) serve as critical biological agents in sustainable agriculture, playing an important role in enhancing nutrient uptake, synthesizing phytohormones, suppressing pathogens, an‎d inducing systemic resistance. This study aimed to molecularly identification of some selec‎ted Bacillus species, including B. amyloliquefaciens, B. megaterium an‎d B. pumilus from the rhizosphere soil of safflower (Carthamus tinctorius L.) treated with these bacteria. Sequences of the 16S rRNA an‎d 23S rRNA genes were downloaded from the NCBI database an‎d aligned using MEGA11 software with the ClustalW algorithm. Species-specific primers (BA23, BM23 an‎d BP23) were designed based on variable regions of the 23S rRNA gene. Primer specificity was eva‎luated using Oligo7, Oligo Analyzer, an‎d Primer-BLAST tools. Rhizosphere soil sampling was conducted at a depth of 10–15 cm, an‎d bacteria were isolated through serial dilution an‎d culturing on Nutrient Agar. Genomic DNA extraction was optimized using the CTAB-phenol-chloroform protocol, an‎d its quality was verified using 0.7% agarose gel electrophoresis. PCR was performed with a temperature gradient of 51–61°C to determine the optimal annealing temperature, an‎d products were sequenced using the Sanger method. Phylogenetic analysis was conducted using the Maximum Likelihood method with partial deletion parameters, employing P. libanensis as the outgroup. Results demonstrated that the designed primers specifically amplified the expected fragments (approximately 600–900 base pairs) without cross-reactivity with non-target DNA. The phylogenetic tree revealed a close relationship between B. amyloliquefaciens an‎d B. subtilis, with a similarity exceeding 99.4%, while the genetic distance from P. libanensis was over 22%. BLASTn analysis confirmed ‎>99% identity, 100% coverage, an‎d an E-value of zero for the target species. However, challenges in identifying B. megaterium were observed due to high genetic similarity within the Bacillus species complex, highlighting limitations of rRNA-based methods. This study provides efficient molecular tools for rapid PGPR identification, with potential applications in biofertilizer production an‎d biological control. The findings underscore the need for complementary markers such as gyrB to overcome taxonomic limitations an‎d suggest that future research should focus on developing Multiplex PCR systems an‎d eva‎luating the impact of climate change on Bacillus populations.

مجيد طالبي , بدرالدين ابراهيم سيدطباطبايي

حميدرضا عشقي زاده

ابوذر سورني , صادق زارعي