توزیع فراوانی و شناسایی مولکولی باکتری‌های بومی محرک رشد در جنوب استان فارس

Background and Objectives: The increasing global population and challenges related to food security have underscored the critical need for sustainable agricultural practices. The overreliance on chemical fertilizers has led to environmental degradation, including soil and water pollution. Plant Growth-Promoting Rhizobacteria (PGPR) offer a promising, eco-friendly alternative by enhancing plant growth through various direct and indirect mechanisms. These mechanisms include improving nutrient availability (e.g., via siderophore production, phosphate solubilization, ammonia production), producing phytohormones like auxins (IAA), and mitigating stress through enzymes like ACC deaminase. However, the effectiveness of PGPR is highly dependent on their adaptation to specific local soil, plant, and climatic conditions. While PGPR potentials are well-established globally, a comprehensive profile of native, multifunctional PGPR in the southern Fars province of Iran remains limited. This study aimed to fill this knowledge gap by 1) isolating and evaluating the frequency distribution of key PGP traits among bacterial isolates from various plant rhizospheres, 2) quantitatively assessing selected isolates for IAA production and ACC deaminase activity, and 3) molecularly identifying the most promising multifunctional bacterial strains.




Materials and Methods: A total of 45 rhizosphere samples were collected from diverse plants (including wheat, barley, alfalfa, lettuce, canola, beetroot, and spinach) in southern Fars province, with geographical coordinates recorded via GPS. From these samples, 181 distinct bacterial isolates were obtained through serial dilution (up to 10⁻⁶) and cultivation on Nutrient Agar, TSA, and King B media. All isolates underwent preliminary qualitative screening for five PGP traits: siderophore production (on CAS agar), ammonia production (in peptone water), phosphate solubilization, potassium release, and zinc solubilization. Based on the results of this screening, 12 superior isolates possessing one or multiple strong PGP traits were selected for further quantitative analysis. The quantitative production of IAA was measured spectrophotometrically using Salkowski's reagent in tryptophan-amended broth. The activity of ACC deaminase was estimated by measuring the amount of α-ketobutyrate produced from ACC. Finally, the molecular identification of the top-performing isolates was carried out by sequencing the 16S rRNA gene using universal primers 27F and 1492R, followed by comparison with sequences in the NCBI database.
 
Results: The initial screening of 181 isolates revealed a high diversity of functional traits within the rhizosphere community of Southern Fars. A total of 94 isolates (52%) exhibited at least one plant growth-promoting trait. The analysis of trait frequency indicated that siderophore production and ammonia production were the most prevalent mechanisms, observed in a significant portion of the population. This high prevalence is likely an adaptive response to the iron-limited, alkaline nature of the calcareous soils in the region. Phosphate solubilization and potassium release were also common, whereas zinc solubilization was less frequent but present in highly efficient strains. Multifunctionality: Venn diagram analysis highlighted that while many isolates possessed single traits, a specific subset demonstrated multifunctionality. From this pool, 12 superior isolates were selected for advanced quantitative characterization. All 12 selected isolates demonstrated the capability to synthesize IAA in the presence of tryptophan, with concentrations ranging significantly. The highest IAA production was recorded for isolates SF1050 (52.10 µg/ml) and SF1078 (51.30 µg/ml), with no significant statistical difference between them. This high level of auxin production suggests a strong potential for these strains to stimulate root elongation and increase root surface area. Regarding stress alleviation traits, ACC deaminase activity was observed in 7 out of the 12 isolates. Isolate SF1044 exhibited the highest enzyme activity (306.93 nmol α-ketobutyrate mg⁻¹ protein h⁻¹), followed by SF1038 (292.07 nmol α-ketobutyrate mg⁻¹ protein h⁻¹). The presence of this enzyme indicates the potential of these strains to facilitate plant growth under biotic and abiotic stress conditions by regulating ethylene levels. 16S rRNA sequencing revealed that the elite isolates belonged to five distinct genera: Bacillus, Pseudomonas, Acinetobacter, Pseudarthrobacter, and Lysinibacillus. Specifically, SF1038 was identified as Bacillus sp., and SF1044, SF1050, and SF1092 were identified as Pseudomonas sp. Other isolates were identified as Acinetobacter sp. (SF1075), Pseudarthrobacter sp. (SF1078), and Lysinibacillus sp. (SF1160). The most significant finding was the identification of Pseudomonas sp. SF1044 as a highly versatile, multifunctional strain possessing all seven tested PGP traits simultaneously, including high IAA production and ACC deaminase activity.




Conclusion: This study provides a comprehensive profile of the frequency distribution of PGP traits among the native rhizobacterial community in southern Fars province. The results confirm the presence of a diverse and potent reservoir of PGPR, with a notable prevalence of nutrient-solubilizing bacteria. The isolation and identification of several highly efficient strains, particularly the multifunctional Pseudomonas sp. SF1044, is a significant outcome. This strain, along with other robust isolates like Bacillus sp. SF1038, presents exceptional potential for development into novel, multi-trait biofertilizers. The use of such native, adapted strains can significantly contribute to sustainable agricultural practices in the region by enhancing crop growth and yield while reducing dependence on chemical inputs. Future research should focus on in-vitro and field-level validation of these promising isolates to formulate effective microbial consortia for regional agricultural application.