Chrome azurol S

Streptomycesisolation and media composition. A total of five soil samples from around healthy plants were collected from 10–20 cm depth of agricultural soil, Qassim University Campus, Buraydah, Qassim, KSA. By the standard serial dilution method, these soil samples were prepared for bacterial strains isolation (Valan Arasu et al. 2009 (link)). Soil samples (3–4 g) of each sample were suspended in distilled water (9 ml) and vortexed. Furthermore, a serial dilution up to 10^‒3 dilution of each sample was performed. Streptomyces were subsequently isolated by spread plate technique on PDA (Potato Dextrose Agar) medium and incubated for a week at 28°C. Selected Streptomyces colonies were isolated and characterized by their colony morphology and pigments. These colonies were further purified and sub-cultured on tryptone soyagar (15 g/l pancreatic digest of casein, 5 g/l enzymatic digest of Soybean, 5 g/l sodium chloride, 15 g/l agar, final pH 7.3). For secondary metabolites production, glucose soybean meal broth (GSB) consisted of 10 g/l glucose, 10 g/l soybean meal, 10 g/l NaCl, 1 g/l CaCO₃, and pH adjusted to 7.0 was used as the production medium.
Isolated strains classification and identification.Morphological characteristics. The morphological properties of isolated Streptomyces strains were characterized with colony characteristics, pigment color, areal hyphae, the opacity of colony, colony consistency, fragmentation pattern, and growth under the surface of liquid media. Otherwise, for visualization of aerial hyphae, hypha, and spore characteristics under the scanning electron microscope (SEM), S. tricolor strain HM10 was grown for 48 h in a growth medium. The bacteria were harvested at 6,000 rpm by centrifugation for 10 min and subjected to the method of a critical drying point (Dhanjal and Cameotra 2010 (link)). The cells were washed with phosphate-buffered saline (PBS, pH 7.4) three times and fixed by incubation in a modified Karnovsky’s fixative solution (2.5 ml of 50% glutaraldehyde, 2 g paraformaldehyde) for four hours. Cells were washed with PBS and distilled water and dehydrated by the increasing ethanol concentrations (30%, 50%, 70%, 90% and 100%) for critical point drying. t-Butyl alcohol was used to layer the dehydrated samples for freeze-drying, subsequently, and the samples were coated with titanium and viewed at 1,000 to 5,000-fold magnification with SEM (AMRAY 3300FE).
Morphological characteristics. The isolated Streptomyces were grown at 28°C for 7 days in Tryptone Soy Agar medium. The soluble pigments color, the hyphae color and airborne hyphae were detected.
PCR amplification of 16S rRNA and phylogenetic characteristics. DNA was extracted according to the simple method of DNA extraction with little modifications (Cook and Meyers 2003 (link)). Briefly, isolated Streptomyces strains were cultured in TSB (tryptone Soy-broth) at 30°C for 24–48 h. Cells were centrifugated for 3 min at 12,000 rpm, washed once with TE buffer (pH 7.7). Cells were resuspended again in TE buffer (500 μl), heated at 95°C for 10 min in boiling water bath, and kept on ice to cool, followed by centrifugation at 12,000 rpm for 5 min. The extracted DNA was transferred to a clean tube and stored at 4°C for PCR amplification. PCR amplification was conducted using GoTaq® Green Master Mix (Promega, USA) for 16S rDNA in 50 μl volumes by universal primers 27 F 5’-AGAGTTTGATCATGGCTCAG-3’ and 1492 R 5’-TACGGTTACCTTGTTACGACTT-3’. PCR products were electrophoresed in 1% agarose gel to ensure the amplification of the fragment of correct size. Products were purified and sequenced (Capillary Electrophoresis Sequencing (CES), ABI 3730xl System, Macrogen company, South Korea). A phylogenetic tree was inferred with a maximum likelihood method using with the following parameters: Tamura-Nei model, Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, Uniform Rates. Evolutionary analyses were conducted in MEGA X (Kumar et al. 2018 (link)).
Antimicrobial activity assays. The isolated Streptomyces strains were grown for three days in GSB liquid media. Their antifungal activity against ten fungal plant pathogens was measured according to Kanini et al. (2013) (link). The fungal strains were grown on Potato Dextrose Agar (PDA) plates for 3 days at 30°C, then a 6-mm mycelium disk from each selected fungus was then placed in the center of a new PDA plate. The bacterial suspensions (50 μl from a 5-day culture of each Streptomyces strain tested) were put into the opposite sides of each PDA plate. The inoculated plates with fungi and Streptomyces were kept in the incubator for five days at 28°C. The antagonistic activity of the strains tested was observed via measuring the inhibition zone distance. The antibacterial assay was also measured with five-day cultures filtrate from Streptomyces tested strains against the bacterial strains selected using the agar well diffusion method with modifications (CLSI 2011 ). Briefly, each tested strain was grown in LB media overnight, and an inoculum of each tested strain (about 2 ml) was added to 25 ml of new LB media before solidification (at nearly 50°C). In the agar medium, wells of six mm in diameter were perforated, and 50 μl of each five-day Streptomyces cultures were placed into the wells, followed by incubation at 30 or 37°C (depended on the bacteria favorite temperature). After 24 h of incubation, the inhibition zones were recorded.
Plant growth promotion (PGP) assessmentin vitro. Three parameters related to plant growth promotion were evaluated in Streptomyces strains.
Siderophores production. The CAS (Chrome Azurol S) assay to detect siderophore production, according to (Schwyn and Neilands 1987 (link)) was applied. Briefly, iron (III) solution was prepared by mixing 1 mM FeCl₃ in 10 ml of 10 mM HCl. In another conical flask, 60.5 mg of CAS was dissolved in distilled water (50 ml). The orange color mixture was then added to the previously prepared solution of the iron (10 ml), which turned the solution color to purple. Whereas stirring, the previous purple solution was slowly poured into HDTMA (hexadecyltrimethylammonium) (72.9 mg), dissolved in 40 ml of distilled water, which turned into dark blue color after mixing. Streptomyces strains on PDB of approximately the same OD₆₀₀ were put into a succinate medium mixed with CAS dye and incubated for 72–96 h. A clear to orange halo around the growing bacterial cells were detected. The molecules’ color intensity and diffusion potential were directly related to the chelating strength and the concentration of produced siderophore.
Production of extracellular indole-3-acetic acid (IAA).Streptomyces strains were grown in nutrient broth medium for one day at 28°C. Cells were diluted up to (10⁸ CFU/ml) in NB medium supplemented with L-tryptophane (500 μg/ml), and grown with shaking for five days at 28°C. Cells were pelleted for 10 min at 12,000 rpm, while the supernatant was collected. Using Salkowski reagent, which consisted of 0.5 M FeCl₃ (1 ml) in 35% HCLO₄ (50 ml), IAA concentration was measured with a colorimetric assay (Bano and Musarrat 2003 (link)) after 25–30 min using a spectrophotometer at the wavelength 530 nm. The standard curve was made to evaluate the IAA concentration.
Phosphate solubilization. Pikovskaya agar (PKV) medium was prepared, and Ca₃(PO₄)₂ was added separately after autoclaving to agar plates. A 50 μl of each strain containing approximately (10⁸ CFU/ml) was added to agar plates and incubated for five days at 28°C. Bacterial colonies with clarification halos around were considered phosphate solubilizers (Donate-Correa et al. 2005 (link)).
Fermentation, extraction, and cancer cell culture. S. tricolor HM10 and Streptomyces thinghirensis strain HM3 were grown in GSB medium for six days. The fermented broth was extracted with equal volume from ethyl acetate, and vacuum evaporated. The resulted extract was dissolved in phosphate buffer saline (PBS, pH 7) and used to assay of cytotoxic activity. The A549 lung cancer cell-lines were purchased from ATCC (VA, USA) and were grown in DMEM according to manufacturer’s instruction. Briefly, A549 cells were grown in DMEM medium with 10% heat-inactivated fetal bovine serum (FBS) at 37°C in 5% CO₂ as described previously (Al Abdulmonem et al. 2020 (link)).
Treatment of lung cancer cells with the twoStreptomycesextracts and cytotoxicity assay. The cultured cancer cells were serum-starved overnight and were treated with S. tricolor HM10 and S. thinghirensis HM3 extracts (10–200 μg/ml) for 12 hours, and the cytotoxicity was determined by the CytoTox-Glo™, Cytotoxicity Assay Kit (Promega, Madison, WI, USA).
DNA sequencing and NCBI Accession Numbers. The 16S rRNA nucleotide sequences for eight Streptomyces strains were deposited in GenBank under the accession numbers MN527229–MN527236.