All media contained 20 g agar and 1 L of seawater, and were adjusted to pH 7.0. For bacterial isolation, 0.05 g L−1 streptomycin and potassium dichromate (50 mL of 1 g L−1 sterilized potassium dichromate in 1 L sterilized media) was added to the
bacterial isolation basic media to inhibit the growth of fungi. For fungal isolation, to inhibit the growth of bacteria, 0.5 g L−1 benzylpenicillin HDAC inhibitor and 0.03 g L−1 Rose bengal were added to the fungal isolation basic media. For bacterial DNA extraction, the selected bacterial isolates were inoculated into 7-mL centrifuge tubes containing 1 mL M2-broth medium (removed 20 g agar from M2) and cultured at 30 °C with shaking at 150 r.p.m. for 3–5 days. Total genomic DNA was extracted from all selected strains as described by Li & De (1995). From the genomic DNA, nearly full-length 16S rRNA gene sequences were amplified by polymerase chain reaction using primers 27°F (5′-GAGTTTGATCCTGGCTCAG-3′)
and 1525R (5′-AGAAAGGAGGTGATCCAGCC-3′; Warneke et al., 2006). All of the primers were synthesized by SBS Genetech (China). The polymerase chain reaction mixtures Selumetinib mw consisted of 12.5 μL Taq premix (TakaRa, China), 1 μL (10 μM) of each primer (TakaRa), 1.5 μL DMSO, 8 μL water and 1 μL of template DNA. After denaturation at 94 °C for 6 min, amplification was performed with 30 cycles of 40 s at 94 °C, 40 s at 53 °C, 2 min at 72 °C and a final extension at 72 °C for 10 min (Lee et al., 2003). Detailed information of fungal DNA extraction and fungal identification are given by Zhang et al. (2012). DNA sequencing of the selected bacterial and fungal isolates was carried out by Invitrogen (China). Sequences were corrected using sequencher, and the most similar sequences in GenBank were found using Basic Local Alignment Search Tool (blast) searches. When the top three matching blast hits were from the same species and
were ≥ 98% similar to the query sequence, this species name was assigned to the selected isolate (Toledo-Hernandez et al., 2008). The antimicrobial activities of bacterial and fungal isolates were determined by a Methocarbamol double-layer technique (Wu et al., 2009). Selected bacterial and fungal isolates were grown on M2 at 30 °C and M7 at 26 °C, respectively, for 5–14 days depending upon the growth rate of the various isolates. Two marine bacteria (Micrococcus luteus and Pseudoaltermonas piscida) and two marine coral pathogenic fungi [A. versicolor (AV) and A. sydowii (AS)] are the indicator microorganisms for the double-layer assay. Detailed information of the antimicrobial activity test is given by Zhang et al. (2012).