Cotton production is negatively affected by both biotic (diseases and insects) and abiotic (high temperature, salinity, water deficit, and extreme pH) factors. Soil-borne diseases, especially wilts and rots, significantly reduce cotton yield. Thus, we aimed to isolate and identify multi-stress tolerant bacterial antagonistic agents (AGAs) against two major soil-borne pathogens, Macrophomina phaseolina and Fusarium oxysporum. A total of 132 isolates with distinct morphologies were recovered from 25 different rhizospheric soil samples of cotton. A dual culture plate and broth assay confirmed the antagonistic activity of the isolates against these phytopathogens. Four selected AGAs thrived in salt stress induced by different NaCl concentrations, up to 1.71 M, except for isolate 62, which survived up to 0.85 M. Under osmotic stress, all the AGAs were tolerant of up to −1.03 MPa. Similarly, all the AGAs were able to survive over a temperature range of 20–50 °C except for isolate 62, which survived up to 45 °C and was regarded as thermotolerant. All four AGAs were able to grow at pH values ranging from 5 to 9. AGA 18 and S46-7 survived under highly acidic conditions (pH 4). These multi-stress tolerant AGAs also exhibited different plant growth-promoting activities, such as mineral solubilization, ACC-deaminase production, and IAA production. Molecular identification revealed the following AGAs: Bacillus siamensis SSVP1 (18), Bacillus halotolerans SSVP2 (34), Pseudomonas aeruginosa SSVP3 (62), and Bacillus tequilensis SSVP4 (S46-7). AGAs with multiple stress tolerance traits can serve as potential biocontrol agents in the field to reduce pesticide consumption in cotton-growing areas.
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