Isolation and Genotypic Identification of Kappa- Carrageenase-Producing Marine Bacteria from "Ice-Ice" Infected Kappaphycus alvarezii (Sacol variety)

Abstract

This study aimed to isolate and identify kappa-carrageenase-producing marine bacteria from Kappaphycus alvarezii (Sacol variety) with "ice-ice" disease. Brown Sacol varieties, showing symptoms (i.e. thalli whitening and softening) were Diseased portions were inoculated on Yeast and Basal Salts medium (Quatrano) plates were further purified through spread-plating. Kappaphycus alvarezii, Green and characterized for "ice-ice" disease collected from Calatagan, Batangas. Extract Tryptone Seawater (YETS) through the swab-streak method and Formation of depression-forming colonies indicated the production of carrageenase. Three categories were recognized according to colony characteristics i.e. color, depression, margin, etc. These groups are 1) peach, represented by QGEnl; 2) large lobate to circular, exhibited by QG3A and YG1 Al; and 3) pinpoint, represented by YG2A1 and QG3B. QGEnl, due to the presence of putative contaminants (white spreaders), was subjected to further purification. Genomic DNA was extracted from YG1AI, YG2A1, QG3A and QG3B using Xanthogenate-SDS method. The 16S rDNA (~1.3 kb) was PCR-amplified from the DNA extracted from the isolates using the primer pair 63F and 1387R with the following PCR conditions for 30 cycles: hot start at 94°C, 3 minutes, denaturation at 94°C, 30 seconds; annealing at 65°C, 30 seconds, elongation at 72°C, 45 seconds; 72°C for 4 minutes. Amplified 16S rDNAs were subjected to Amplified Ribosomal DNA Restriction Analysis (ARDRA) with restriction enzymes Mspl, Rsal and HaeWX to determine similar isolates based on restriction profiles, which showed that YG1 Al and QG3A are similar isolates, as well as YG2A1 and QG3B. The purified PCR products of 〜1.3 kb and 20ng/|iL concentration were sent to the Davis Sequencing Facility for sequencing. Analysis of the forward and contig sequences of the samples through Basic Local Alignment Search Tool (BLAST) and Sequence Match (SeqMatch) showed that the closest identity of YG1 Al and QG3A is Microbulbifer sp. with a bit score of 2323 using BLAST and a 98.8% similarity using SeqMatch. On the other hand, the closest matches of YG2A1 and QG3B are Bacterium CWISO12 with a bit score of 2466 using BLAST and a 99.7% similarity using SeqMatch, and Vibrio sp. that obtained a bit score of 2460 and a 99.8% similarity using SeqMatch. However, the phylogenetic tree showed that the closest phylogenetic neighbor of both YG1 Al and YG2A1 is the Vibrio - Photobacterium group.