The study was conducted at three different sites of southern part of Gulf of Kachchh, Gujarat. The geographical location of collection sites are: Station 1―Sikka (Latitude 22˚26.406'N Longitude 069˚50.029'E) (coastal wetland, port activity), Station 2―Valsura (Latitude 22˚33.578'N Longitude 070˚02.502'E) (coastal wetland, ship breaking activity) and Station 3―Khijadiya (Latitude 22˚31.375'N Longitude 070˚08.099'E) (inland wetland, bird sanctuary) (see
About 1g of rhizosphere soil of Avicennia marina was transferred to 50 mL test tube containing 10 mL sterile distilled water and vortexed vigorously for 10 min. The resulting solution containing the rhizosphere bacteria was serially diluted up to 10−4 using sterile distilled water. 100 µl aliquot was taken from each dilution and plated in triplicate onto Zobell’s marine agar 2216 (ZB agar) (Himedia, India), MPM (Synthetic sea water media) agar medium [
The morphological characterization of the bacterial colonies were carried out on the basis of their shape, size, colour, margin, elevation on the media and Gram staining were performed to decide the further determinative protocol.
The pure culture were subjected to identification by Bergey’s Manual of Determinative Bacteriology (Volume-4)
using catalase test with 3% hydrogen peroxide, Anaerobic Test, Oxidative Fermentative (O/F test), methyl red test, Voges Proskaeur test, glucose test, sorbitol test and mannitol test.
Genomic DNA Isolation
Culture was centrifuged at 10,000 rpm for 5 min. Pellet was collected and resuspended by adding 9 ml of STE buffer (0.1 mM NaCl, 10 mM Tris, 10 mM EDTA) 1 ml of SDS (10% Stock Solution). The suspension was incubated at 70˚C for 1 hr. and centrifuged at 6000 rpm for 10 min at room temperature. The supernatant was collected in fresh tube and add equal volume of Phenol:Chloroform:Isoamyl alcohol (PCI mix) (25:24:1) was added and mixed slowly. The suspension was centrifuged at 6000 rpm for 10 min. The aqueous phase in fresh tube. Equal vol. of Chloroform: Isoamyl alcohol (24:1) and mix slowly and centrifuged at 6000 rpm for 10 min. The aqueous phase was collected and added double the vol. of absolute alcohol was added. The tube was subjected to overnight incubation in −20˚C. The solution was centrifuged at 6000 rpm 4˚C for 10 min and the pellet was resuspended in 1/10th ml of 3M sodium acetate and 10 ml of absolute alcohol and centrifuged at 6000 rpm 4˚C for 10 min. The supernatant was discarded and the pellet was air dried. The pellet was dissolved in 1 ml sterile TE buffer. The DNA quality was checked using Agarose gel electrophoresis and quantified using Nanodrop.
PCR Amplification and Phylogenetic Analysis
The 16S rRNA gene from the genomic DNA was amplified using primers F 5’-AGAGTTTGATCCTGG CTAG-3’, R 5’-CGGTTACCTTGTTACGACTT-3’ and F5’-TGGAGAGTTTGATCCTGGCTCAG-3’, R 5’- GGTTACCTTGTTACGACTT-3’ (at Xcelris Genomics Pvt. Ltd., Ahmedabad and Junagadh Agricultural University, Junagadh). The PCR reaction mixture (25 µl) comprised of bacterial DNA (50 ng), 10 pmol each of the two oligonucleotide primers, 2.5 mM of each deoxynucleoside triphosphate, 1.5 U of Taq polymerase and 2.5 µl of 10× buffer. The amplification was performed using following PCR cycle: the initial denaturation at 94˚C for 5 min, followed by 35 cycles of 94˚C for 30 s, 60˚C for 30 s and 72˚C for 90 s and a final extension of 72˚C for 10 min. PCR product was electrophoresed on 1.5% agarose gel. PCR products varied from 1200 bp to 1500 bp. Sequencing was performed by ABI 3730xl Genetic Analyzer 96 well capillary system.FASTA files were subjected to BLAST for further analysis [
Identification utilizing GenBank database was carried out by internet-based 16S rRNA gene sequence comparison software utilizing the basic local alignment search tool with default settings. The closest species level match (% identity) was considered the identification.