Introduction

The increasing global population and the limited availability of natural resources, particularly fertile soils, have posed significant challenges to modern agriculture in achieving sustainable food production. During their growth, plants are in constant and direct contact with microorganisms present in the root vicinity, collectively known as the rhizosphere. Microorganisms inhabiting the rhizosphere of various plants exert multiple positive effects on their hosts through diverse mechanisms and are commonly referred to as plant growth-promoting rhizobacteria (PGPR). In the rhizosphere, plant roots release exudates that serve as attractants for microorganisms, ultimately enhancing the physical and chemical properties of the surrounding soil. These exudates also play a crucial role in maintaining the structure and function of microbial communities near the plant roots. PGPR promote plant growth through various biological mechanisms, including atmospheric nitrogen fixation, siderophore production, phytohormone synthesis (such as auxins, gibberellins, and cytokinins), and the solubilization of insoluble phosphorus compounds. Furthermore, certain bacteria enhance the availability of essential nutrients to plants. Research has demonstrated that the application of these bacteria can increase plant tolerance to abiotic stresses, such as salinity and drought. Moreover, PGPR can indirectly bolster plant resistance to biotic stresses by inducing systemic resistance, exerting antibiotic effects, and potentially enhancing the plant's cellular metabolite content. By reducing the reliance on chemical fertilizers and pesticides, PGPR also contribute to soil health and environmental sustainability.
In general, the development and application of biological products based on plant growth-promoting rhizobacteria (PGPR), as an alternative or supplement to chemical fertilizers and pesticides, have gained significant attention in agricultural research and applications. This approach is recognized as an efficient and environmentally sustainable method for enhancing crop productivity. Therefore, this study was conducted to evaluate the biological capabilities of certain plant growth-promoting bacterial isolates.
Materials and methods
Bacterial isolates effective in promoting plant growth were obtained from various genera through the Soil and Water Research Institute of Iran and the University of Jiroft. A total of 26 bacterial isolates were collected, including 15 isolates belonging to the genus Bacillus, 5 to Pseudomonas, and 3 each to the genera Azotobacter and Azospirillum. To ensure the purity of the isolates, all 26 were re-cultured using the pentagonal streaking method on NAS medium. Additionally, their morphological and phenotypic characteristics were examined using Gram staining, catalase testing, and evaluation of levan production capability.
Siderophore production was assessed on CAS-Agar medium following the method described by Alexander and Zuberer. Phosphate solubilization ability was tested using Sperber’s method, which involves a medium containing tricalcium phosphate that causes turbidity. The formation of a clear halo around the bacterial colonies was considered an indication of phosphate solubilization. For nitrogen fixation assessment, a nitrogen-free Winogradsky medium was prepared. To identify nitrogen-fixing bacteria, 5 µL of the bacterial suspension was spot-inoculated onto the medium and incubated at 28 °C for seven days. Bacterial growth or lack thereof was compared to that on NAS medium. Qualitative assessment of IAA (indole-3-acetic acid) production was performed using the method described by Bano and Musarrat. Bacterial isolates were cultured in 100 mL of liquid LB medium and incubated at 28 °C for 72 hours in a shaker incubator at 120 rpm. The cultures were then centrifuged at 5000 rpm for 30 minutes, and 2 mL of the supernatant was mixed with 1 mL of Salkowski reagent. The appearance of a pink to reddish color indicated IAA production.
Results and discussion
The results of the analysis of variance (ANOVA) for the ability of the isolates to produce siderophores showed significant differences at the 1% probability level among some of the isolates. The results of the mean comparison indicated that 15 isolates were capable of producing siderophores, with the highest production observed in the isolates UJB33 (Bacillus subtilis), UJB22 (Bacillus paralicheniformis), UJB31 (Pseudomonas fluorescens), UJB32 (Azotobacter chroococcum), and UJB36 (Azotobacter salinestris). The lowest production was observed in the isolates UJB24 and UJB34, which belonged to the genera Pseudomonas and Bacillus, respectively.
The ANOVA results for the semi-quantitative phosphate solubilization assay revealed significant differences at the 1% probability level among the isolates. Furthermore, the results of the mean comparison indicated that the highest phosphate solubilization was observed in the isolates UJB32, UJB11, UJB24, and UJB33, with halo diameters of 11.33, 9.33, 7.66, and 5.66 mm, respectively. The lowest phosphate solubilization was observed in the isolates UJB19, UJB28, and UJB18, with halo diameters of 2.66 and 2.33 mm. The qualitative assessment of nitrogen fixation by the antagonistic isolates revealed that 19 isolates were capable of growth in nitrogen-free medium, indicating their nitrogen-fixing ability. These isolates belonged to the genera Bacillus, Pseudomonas, Azotobacter, and Azospirillum. Growth and non-growth of the isolates were compared with the nutrient agar (NA) medium. The results of the qualitative assessment of auxin production by the isolates showed that 10 of the tested isolates were capable of producing auxins in the presence of tryptophan.
Conclusion
The results of this study demonstrated that isolates UJB33, UJB22, UJB31, UJB32, UJB36, UJB12, UJB18, UJB28, UJB11, UJB25, UJB29, UJB15, UJB35, UJB24, and UJB34 were capable of producing siderophores. These isolates belonged to four bacterial genera: Bacillus, Azotobacter, Azospirillum, and Pseudomonas. It was also found that isolates UJB32, UJB11, UJB24, UJB33, UJB20, UJB26, UJB31, UJB34, UJB36, UJB25, UJB35, UJB16, UJB18, UJB28, and UJB19 were able to solubilize phosphate. Phosphate solubilization by bacterial isolates is an important trait that can enhance plant growth and yield by improving phosphorus availability. Moreover, the ability to produce indole-3-acetic acid (IAA) in the presence of tryptophan was confirmed in isolates UJB11, UJB29, UJB12, UJB15, UJB32, UJB34, UJB27, UJB25, UJB14, and UJB18. IAA is a key phytohormone that promotes plant growth and contributes to increased plant height. In addition, nitrogen fixation tests showed that isolates UJB31, UJB32, UJB36, UJB12, UJB18, UJB11, UJB26, UJB25, UJB29, UJB15, UJB34, UJB35, UJB30, UJB14, UJB19, UJB17, UJB20, UJB21, and UJB16 were capable of fixing atmospheric nitrogen. These isolates also belonged to the genera Bacillus, Azotobacter, Azospirillum, and Pseudomonas. Overall, this study highlights the potential of plant growth-promoting bacteria (PGPB) from the genera Pseudomonas, Bacillus, Azotobacter, and Azospirillum to improve plant growth and optimize soil resource utilization through multiple mechanisms, including IAA production, nitrogen fixation, phosphate solubilization, and siderophore production. These bacteria play a vital role in enhancing nutrient availability to plants and suppressing soil-borne pathogens. Their application as biofertilizers offers a sustainable alternative to chemical fertilizers and holds promise for improving crop productivity in modern agriculture.