Characterization of bacterial endophytes isolated from Cannabis sativa L. and Chelidonium majus L. for their application as biostimulants and biocontrol agents.

Endophytic bacteria contribute to plant growth, stress tolerance, and pathogen resistance. Their effective use in agriculture requires the identification of strains that combine multiple beneficial traits with consistent performance across different field conditions. Accordingly, this study examines Bacillus and Pseudomonas endophytes isolated from Cannabis sativa L. and Chelidonium majus L. for plant growth promotion, abiotic stress tolerance, and biocontrol properties. Plant growth-promotion traits included indole, siderophore, and organic acid production, phosphate and zinc solubilization, and biofilm formation. Results showed that all the tested bacterial isolates produced indoles, with the highest levels recorded in Pseudomonas strain PPW-26, whereas several Pseudomonas strains exhibited strong siderophore production. Strain PPW-26 tested positive for methyl-red, indicating organic acid production, whereas other Pseudomonas strains tested negative. Moderate to high nutrient solubilization profiles were observed across all Pseudomonas strains. Bacillus strains, particularly BS-114, exhibited higher biofilm formation relative to Pseudomonas. Assessment of abiotic stress tolerance included proline accumulation, superoxide dismutase activity, and growth under varying temperature, salinity, and drought conditions. All strains displayed tolerance to the tested stresses, with Bacillus strains showing stronger resilience to high temperature and salinity, accompanied by elevated proline accumulation and superoxide dismutase activity in selected strains. Biocontrol potential was evaluated through biosurfactant production and antifungal activity. Bacillus strains showed high biosurfactant activity and strong inhibition of fungal pathogens. Strain BS-120 exhibited broad-spectrum inhibition against Fusarium oxysporum, Fusarium graminearum, and Rhizoctonia solani-AG3. Analysis of genome sequences identified biosynthetic gene clusters encoding antifungal metabolites, including fengycin and surfactin, consistent with the observed inhibition. Genome-wide similarity analysis and ANI-based clustering revealed the presence of highly similar and genetically distant strains within each genus. For Bacillus spp., ANI values ranged from 87.62% to 98.83%, whereas for Pseudomonas spp. they ranged between 83.91% and 99.99%, confirming the presence of substantial intra-genus diversity. Phylogenetic analysis showed well-supported clades consistent with ANI clustering. Overall, this study demonstrates that endophytic Bacillus and Pseudomonas strains exhibit complementary and strain-dependent traits associated with plant growth promotion, stress tolerance, and pathogen suppression, supporting their further evaluation as potential bioinoculants for sustainable agriculture.