Functional and Statistical Validation of Nitrogen-Fixing Plant Growth–Promoting Bacteria for Ethylene Regulation and Seed Germination Under Osmotic Stress

Background: Plant growth-promoting bacteria (PGPB) enhance plant productivity through multiple mechanisms, including biological nitrogen fixation, ammonia production, ACC deaminase activity, and improved tolerance to abiotic stresses. The present study aimed to evaluate selected bacterial isolates for their plant growth-promoting characteristics and assess their ability to mitigate drought stress during seed germination, with the objective of identifying promising candidates for biofertilizer development. Results: Among the tested isolates, Corynebacterium accolens, Bacillus rugosus, and Lactobacillus pasteurii DSM 23907 exhibited strong ammonia production, indicating high nitrogen-fixing potential. ACC deaminase screening confirmed α-ketobutyrate production in several isolates, demonstrating enzymatic activity. One-way ANOVA revealed significant differences in ACC deaminase activity among treatments (p < 0.05), with Lactobacillus pasteurii DSM 23907 showing the highest activity among the bacterial isolates (1.487 ± 0.050), followed by Bacillus rugosus , while Corynebacterium accolens and Cytobacillus firmus exhibited significantly lower but statistically similar activities. The acetylene reduction assay further confirmed nitrogenase activity in all isolates, with Lactobacillus pasteurii DSM 23907 recording the highest activity among the tested bacteria (38.46 ± 3.59 nmol/OD/hr), significantly exceeding the other isolates (F(4,10) = 72.6, p < 0.0001). Seed germination assays demonstrated that PEG-induced drought stress significantly reduced germination percentage, delayed germination, inhibited root and shoot growth, and decreased seedling vigor. However, bacterial inoculation markedly alleviated these adverse effects by improving germination (76.0–77.3%), reducing mean germination time, enhancing seedling growth, and more than doubling the Seedling Vigor Index compared with uninoculated stressed seedlings. One-way ANOVA confirmed significant treatment effects for all measured growth parameters (p < 0.001), while heatmap analysis further illustrated the ability of bacterial inoculation to partially restore seedling performance under drought stress. Conclusions: The investigated bacterial isolates exhibited multiple plant growth-promoting traits, including ammonia production, ACC deaminase activity, and nitrogenase activity, with Lactobacillus pasteurii DSM 23907 demonstrating the strongest overall performance, followed by Bacillus rugosus . Bacterial inoculation significantly improved seed germination, seedling growth, and drought tolerance under PEG-induced osmotic stress, highlighting the potential of these isolates as multifunctional biofertilizers for sustainable agriculture and crop production under water-limited conditions.