Tomato (Solanum lycopersicum L.) holds significant importance as a primary vegetable crop globally. In this research, the extracellular green-synthesis of silver nanoparticles was accomplished by using some Gram positive bacteria. Three isolates including KM59, SM88, and KM93 exhibited the highest nanoparticle biosynthesis activity. The isolates were identified according to colony morphology and the 16S rRNA gene sequence identities, as Streptomyces spp. The obtained AgNPs were characterized by TEM and exhibited various shapes, including hexagonal, pentagonal, spherical, and triangular. The MIC for Ralstonia solanacearum was determined to be 25, 6.25, and 25 μg/mL for the AgNPs93, AgNPs88, and AgNPs59, respectively. The MIC of AgNPs93, AgNPs88, and AgNPs59 against Xanthomonas campestris was assessed at concentrations of 25, 12.5, and 50 μg/mL, respectively. In the case of Alternaia alternata, the MIC for AgNPs93 and AgNPs59 was found to be 50 μg/mL, while for AgNPs88, it was 25 μg/mL. Furthermore, the MIC of AgNPs88 against Rhizoctonia solani was determined to be 12.5 μg/mL, whereas for AgNPs93, it was 25 μg/mL, and for AgNPs59, it was 50 μg/mL. Disease index was reduced (AgNPs88 demonstrated the most potent inhibitory effect, whereas AgNPs59 displayed the least inhibitory effect) when tomato plants inoculated with fungal and bacterial pathogens treated with the AgNPs at MIC concentrations. The cytotoxicity of AgNPs on the B16F10 cancer cell lines determined by the means of MTT assay. Biosynthesis of AgNPs using Actinobacteria is an eco-friendly process. In agriculture, medical sciences, and industry, process optimization can become a valuable technology for mass production.

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