Combined application of melatonin and sp. strain IPR-4 ameliorates drought stress tolerance via hormonal, antioxidant, and physiomolecular signaling in soybean.

The role of melatonin and plant growth-promoting rhizobacteria (PGPR) in enhancing abiotic stress tolerance has been widely investigated. However, the mechanism underlying the interaction between melatonin and PGPR in drought stress tolerance is poorly understood. In this study, we investigated the role of sp. strain IPR-4 co-inoculated with melatonin (IPR-4/MET) to ameliorate drought stress response in soybean. Initially, 16 random isolates were selected from a previously pooled collection of isolates from soil at plant physiology lab, and were screesn for plant growth promoting (PGP) traits and their survival rate polyethylene glycol (PEG6000) (5%, 10%, and 15%). Among these isolate Bacillus sp. strain IPR-4 were selected on base of its significant PGP traits such as the survival rate gradient concentrations of PEG6000 (5%, 10%, and 15%) compared to other isolates, and produced high levels of indole-3-acetic acid and organic acids, coupled with exopolysaccharide, siderophores, and phosphate solubilization under drought stress. The Bacillus sp. strain IPR-4 were then validated using 16S rRNA sequencing. To further investigate the growth-promoting ability of the sp. IPR-4 and its potential interaction with MET, the bacterial inoculum (40 mL of 4.5 × 10 cells/mL) was applied alone or in combination with MET to soybean plants for 5 days. Then, pre-inoculated soybean plants were subjected to drought stress conditions for 9 days by withholding water under greenhouse conditions. Furthermore, when IPR-4/MET was applied to plants subjected to drought stress, a significant increase in plant height (33.3%) and biomass (fresh weight) was observed. Similarly, total chlorophyll content increased by 37.1%, whereas the activity of peroxidase, catalase, ascorbate peroxidase, superoxide dismutase, and glutathione reductase increased by 38.4%, 34.14%, 76.8%, 69.8%, and 31.6%, respectively. Moreover, the hydrogen peroxide content and malondialdehyde decreased by 37.3% and 30% in drought-stressed plants treated with IPR-4 and melatonin. Regarding the 2,2-diphenyl-1-picrylhydrazyl activity and total phenolic content, shows 38% and 49.6% increase, respectively. Likewise, melatonin-treated plants enhanced the uptake of magnesium, calcium, and potassium by 31.2%, 50.7%, and 30.5%, respectively. Under the same conditions, the salicylic acid content increased by 29.1%, whereas a decreasing abscisic acid content (25.5%) was observed. The expression levels of , , and were recorded as the lowest. However, -melatonin-treated plants recorded the highest expression levels (upregulated) of and , , and in response to drought stress. In a nutshell, these data confirm that sp. IPR-4 and melatonin co-inoculation has the highest plant growth-promoting efficiency under both normal and drought stress conditions. sp. IPR-4/melatonin is therefore proposed as an effective plant growth regulator that optimizes nutrient uptake, modulates redox homeostasis, and enhances drought tolerance in soybean plants.