Multi-stress tolerant Bacillus from salt-affected arid soil

Salinity, drought, and heat severely constrain microbial activity in arid soils. This study investigated whether multi-stress tolerance in Bacillus isolated from the rhizosphere of Suaeda vera is associated with a genomic complement of stress-response and plant growth-promoting genes. We isolated 150 bacterial isolates from this rhizosphere soil at saline sites (16–32 dS/m) of Sehb El Mesjoune, central Morocco. Among these isolates, 65 were classified as multi-stress tolerant, as they grew in culture media under salinity (1.7–3.4 M NaCl), drought (10–20% PEG), and elevated temperatures (≥47°C), with Bacillus (33%) as the dominant genus. Out of these isolates, five Bacillus strains were selected for further analysis. Whole-genome sequencing revealed a genomic architecture with multiple abiotic stress tolerance mechanisms, including KtrABCD transporters, Na+/H+ antiporters, the opuABCD pathway, and the stressosome sensory complex, with operons for biofilm formation (epsA-O, tasA, bslA) potentially relevant to root association. Comparative ortholog analysis detected 444 orthogroups common to the strains from Sehb El Mesjoune and the saline reference B. halotolerans ZB201702 but absent from the non-saline reference B. velezensis FZB42. Of these, 81 were annotated as stress tolerance or plant growth-promoting genes, a pattern that may reflect environmental filtering, although species-level ancestry cannot be excluded. Additionally, in vitro assays and SEM imaging confirmed these traits were functionally expressed, resulting in Bacillus strains attaching to wheat seeds. These findings suggest that the five selected Bacillus strains represent promising candidates for future greenhouse and field testing as bacterial inoculants for crops grown in salt-affected arid soils.