Biogenic synthesis of silver nanoparticles using Bacillus cultures and their antibacterial activity against phytopathogenic Pseudomonas syringae
DOI:
https://doi.org/10.15407/hftp17.03.378Keywords:
“green” synthesis, silver nanoparticles, cell-free extract, Bacillus subtilis, Bacillus cereus, electrokinetic potential, antimicrobial properties, Pseudomonas syringaeAbstract
Bacteria of the Bacillus genus are known in agroecology as a powerful tool for biocontrol due to their ability to produce secondary metabolites that have a suppressive effect on many phytopathogens. This work demonstrates an environmentally friendly approach to the synthesis of silver nanoparticles (NPs) using cell-free extracts (CE) of Bacillus subtilis B1239 and Bacillus cereus VKPM 5059 bacteria from nutrient media of various compositions. Silver NPs have been obtained by biogenic synthesis using CE of soil bacteria B. subtilis and B. cereus. According to the data obtained, CE B. subtilis-NB, CE B. subtilis-9M, and CE B. cereus-NB were acted as reducing, stabilizing, and modifying agents in the process of AgNPs synthesis, which took place at room temperature by photo-stimulated pH-dependent reduction. The NPs were studied using spectroscopy in the UV-visible range, dynamic light scattering (DLS) method, microelectrophoresis and electron microscopy (TEM images and selected area electron diffraction SAED). The biosafety of AgНЧ samples in vitro was determined by the parameters of cyto- and genotoxicity using eukaryotic cells of the L929 line. Antimicrobial activity was evaluated using the disc-diffusion method and by counting Pseudomonas syringae bacteria in sterile soil contaminated with this phytopathogen and treated by the studied AgNP samples. The effect on the indigenous microflora of the soil was evaluated by adding AgNPs in the defined bactericidal concentration to non-sterile soil and counting the total number of soil microorganisms. During the formation of AgNPs, the important role of the ratio of precursor concentration and CE, as well as the pH value, in ensuring their high stability was elucidated. It was shown that bacterial metabolites in CE are responsible for the size formation, high electrokinetic potential, and the peculiarities of the effect of NPs on microorganisms. The formation of the most stable AgNPs occurred at pH 9 and silver concentrations of 1.0?2.0 mM in CE B. subtilis-NB (dilution 1:6), and 1.0 mM silver in CE B. subtilis-9M and CE B. cereus-NB (1:6). Under these conditions, the particles had high electrokinetic potential and small size, which ensured their stability. A pronounced antibacterial effect of biogenic AgNPs against the known phytopathogen of corn diseases P. syringae was found in the concentration range of 30.8–107.9 ?g/ml. Among the studied nanopreparations, biogenic AgNPs formed in CE B. subtilis-NB showed the highest antimicrobial effect, along with the lowest cytotoxicity and absence of genotoxicity for eukaryotic cells. At the same time, a slight degree of suppression of the indigenous soil microflora was observed under these NPs influence, with a transition to a stimulating effect on the 14th day, indicating their high selectivity. The fulfilled studies have demonstrated the potential of using the synthesized AgNPs based on CE B. subtilis for further application in agroecology and plant growing as effective agents for combating plant diseases and protecting soil health.
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