Streptomyces isolation and media composition.
A total of five soil samples were collected from10-20 cm depth of cultivated lands, Qassim region, Saudi Arabia. These soil samples were prepared for isolation of bacterial strains by the standard serial dilution method (Valan Arasu, et al. 2009). Rhizospheric soil samples (4–5 g for each) were suspended in 9 ml of distilled water and vortexed. Then, serial dilutions of each sample were carried out up to10−3dilution. Actinobacteria were subsequently isolated by spread plate technique of serial dilution of soil samples on PDA (Potato Dextrose Agar) medium and incubated at 28 ºC for a week. Selected Streptomyces colonies were isolated and characterized by their colony morphology and pigments. These colonies were further purified and sub-cultured on tryptone soya agar ((g/l) Pancreatic digest of casein 15, Enzymatic digest of soya bean 5, Sodium chloride 5, Agar 15, Final pH 7.3). For secondary metabolites production, Glucose Soya bean meal Broth (GSB) ((g/l) Glucose 10.0, Soya bean meal 10.0, NaCl 10.0, CaCO3 1.0 and pH adjusted to 7.0) was used as the production medium.
Classification and identification of strains
Observation of morphological characteristics
The morphological properties of isolated Streptomyces strains were characterized such as colony characteristics, pigment color, areal hyphae, opacity of colony, colony consistency, fragmentation pattern and growth under surface of liquid media. For aerial hyphae and hypha characteristics in scanning electron microscope (SEM), Actino12 strain was grown for 48 h in growth medium. Harvested cells by centrifugation at 6.000 rpm for 10 min were processed via a critical point drying method (Dhanjal and Cameotra 2010); With phosphate buffered saline (PBS, pH 7.4), the cells were washed three times and fixed by incubation in modified Karnovsky’s fixative solution (2.5 ml of 50% Glutaraldehyde, 2 gm Paraformaldehyde) for four hours. Again, cells were washed with PBS, followed by a distilled water wash and dehydrated by critical point drying through a series of alcohol dehydration steps (30%, 50%, 70%, 90% and 100%). The dehydrated samples were layered with t-butyl alcohol for freeze drying, sputter coated with titanium and viewed at 1,000 to 5,000 X magnification on a scanning electron microscope (AMRAY 3300FE).
- Physiological and biochemical characteristics
The isolated Streptomyces were cultured at 28 °C for 7 days in Tryptone Soya Agar medium. The soluble pigments color, the hyphae color and airborne hyphae were detected in Glucose Soya bean meal agar.
- PCR amplification of 16S rRNA and Phylogenetic relationships
DNA was extracted according to the simple method of DNA extraction with little modifications (Cook and Meyers 2003). Isolated Streptomyces strains were grown in TSB (tryptone soya broth) at 30 ºC for 48 h. Cells were collected with centrifugation at 12.000 rpm for 3 min. and washed once with TE buffer (pH 7.7). Cells were resuspended again in 0.5 ml TE buffer, heated in boiling water bath for 10 min and allowed to cool then centrifuged (12000 rpm for 5 min). 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′- AGA GTT TGA TCA TGG CTC AG-3 and 1492 R 5′-TACG GTT ACC TTG TTA CGA CTT-3. PCR products were electrophoresed on 1% agarose gels to ensure the fragment of the correct size had been amplified. Products were purified and sequences (Capillary Electrophoresis Sequencing (CES), ABI 3730xl System, Macrogen Company, South Korea). A phylogenetic tree was inferred with a maximum likelihood method with following parameter: Tamura-Nei model, Neighbor-Joining method for initial tree, Bootstrap method approach, Uniform Rates. Evolutionary analyses were conducted in MEGA X (Kumar, et al. 2018).
Antimicrobial Activity Assays
Isolated Streptomyces strains were grown for 3 days in GSB liquid media. Antifungal activities against 10 fungal plant pathogens were measured according to (Kanini, et al. 2013). For the previous assay, potato dextrose agar (PDA) plates were used for the fungal strains grown for 3 days at 30 ºC, then a 6 mm mycelium disk from each selected phytopathogenic fungi were then placed in a new PDA plate center. On the other hand, bacterial inoculations were put into the opposite sides of each PDA plate, containing 50 µl of a 5-day culture from each tested Streptomyces strain. The inoculated plates with fungi and Streptomyces were incubated for five days at 28 °C. The antagonistic activity was observed via measuring the inhibition zone distance. On the other side, antibacterial assay was measured via five-day cultures filtrates from grown Streptomyces tested strain against selected bacteria by using the Kirby-Bauer agar well-diffusion method with modifications (Institute 2011), Briefly, each tested strain was grown overnight in LB media, an inoculum from each tested strain (about 2 ml) were added to 25 ml from new LB media before solid (at nearly 50 ºC). Six mm (diameter) wells were perforated in the agar medium, and 50 µl of each 5- days Streptomyces culture were placed into the well and incubated at 30 or 37 ºC (according to the best temperature for each bacteria). Inhibition zones were measured after 24 h of incubation.
In vitro Assessment of Plant Growth Promotion (PGP) Traits in Streptomyces
Three parameters related to plant growth promotion were evaluated in isolated Streptomyces strains.
The CAS (Chrome Azurol S) general assay to detect siderophore production according to (Schwyn and Neilands 1987) was applied. 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 into purple. While stirring, the previous purple solution was slowly poured into HDTMA (hexadecyltrimethylammonium) (72.9 mg) dissolved in 40 ml of distilled water which turned the color into blue dark after mixing. Growing Streptomyces cells on PDA liquid medium were taken with approximately the same OD600 and put into succinate medium mixed with CAS dye and incubated for 72-96 h. A clear to orange halo around growing cells were detected. Color intensity and size of detected halos is directly related to chelating strength and the amount of produced siderophore.
- Extra cellular indole-3-acetic acid (IAA) production
Isolated Streptomyces strains were grown in nutrient broth medium for one day of incubation at 28 ºC. Cells were diluted up to (108 cfu/ml) in NB medium supplemented with L-tryptophane (500 µg/ml) and grown with shaking for 5 days at 28 ºC. Cells were centrifuged at 12,000 rpm for 10 min and the supernatant was collected. Using Salkowski reagent (1 ml of 0.5 FeCl3 in 50 ml of 35% HCLO4), IAA concentration was measured by colorimetric assay (Bano and Musarrat 2003) and the pink color of individual assays was measured after 25-30 min using spectrophotometer at 530 nm. Standard curve was achieved to evaluate the IAA concentration.
Pikovskaya agar (PKV) medium were 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 5 days at 28 ºC. Bacterial colonies with clarification halos around them were considered as phosphate solubilizers (Donate-Correa, et al. 2005).
DNA Sequencing and Nucleotide Sequence Accession Numbers
The 16S rRNA nucleotide sequences for selected eight Streptomyces strains were achieved and deposited in GenBank under the accession numbers MN527229 - MN527236.