Comparative Analysis of Rhizobacterial Isolates from Peanut (Arachis hypogaea L.) with Potential as Plant Growth-Promoting Rhizobacteria (PGPR) Based on 16S rRNA Identification

Abstract

Plant Growth-Promoting Rhizobacteria (PGPR) are beneficial bacteria that colonize the rhizosphere and enhance plant growth through nutrient provision and phytohormone production. This study aimed to isolate bacteria from root nodules of peanut (Arachis hypogaea L.) and evaluate their potential as PGPR. Bacteria were isolated from peanut root nodules, followed by qualitative screening of PGPR traits, including nitrogen fixation, indole-3-acetic acid (IAA) production, phosphate solubilization, and protease activity. Selected isolates were further identified molecularly using the 16S rRNA gene. A total of 16 isolates were obtained, consisting of 12 Gram-negative and 4 Gram-positive bacteria with diverse colony morphologies. PGPR screening showed that 6 isolates were capable of nitrogen fixation, 8 produced IAA, all isolates solubilized phosphate on Pikovskaya medium, and 10 exhibited protease activity. Based on these traits, five representative isolates (AH2, AH5, AH9, AH11, and AH14) were selected for molecular identification. BLAST analysis revealed that the isolates were closely related to Ralstonia sp. (AH2 and AH14), Ralstonia sp. T21-1 (AH11), Burkholderia stabilis (AH5), and Martinezella tropici strain SARCC-715 (AH9). Phylogenetic analysis confirmed that isolate AH9 clustered within the Rhizobium clade, showing 97.28% sequence similarity and strong bootstrap support (100%). These results indicate that peanut root nodules harbor diverse bacteria with plant growth-promoting traits. Furthermore, the identification of Ralstonia and Burkholderia isolates raises biosafety concerns, rendering them unsuitable as biofertilizer candidates without extensive safety evaluations. Therefore, only isolate AH9 (closely related to Martinezella tropici) represents a promising and safer candidate for development as a biofertilizer for peanut cultivation. The in planta validation test shows that isolate AH9 is significantly effective in increasing peanut plant growth based on parameters such as number of leaves, plant height, and dry and fresh weight.