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.
