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Coral Diseases in Panjang Island, Java Sea: Diversity of Anti–Pathogenic

Bacterial Coral Symbionts

Article · December 2015

DOI: 10.1016/j.proche.2015.03.004

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Procedia Chemistry 14 (2015) 15 – 21

2nd Humboldt Kolleg in conjunction with International Conference on Natural Sciences,

HK-ICONS 2014

Coral Diseases in Panjang Island, Java Sea:

Diversity of Anti–Pathogenic Bacterial Coral Symbionts

Agus Sabdonoa,b, Paiga Hanurin Sawonuaa, Ary Giri Dwi Kartikaa,

Jasmine Masyitha Ameliaa, Ocky Karna Radjasaa,b*
a
Magister of Marine Science, FPIK, Diponegoro University, Jl. Prof. H. Soedarto, Semarang 50275, Indonesia
b
Tropical Marine Biotechnology Research Center, Diponegoro University, Jl. Prof. H. Soedarto, Semarang 50275, Indonesia

Abstract

Corals are experiencing severe decline due to coral diseases. The main aim of this paper was to screen anti-pathogenic
property of coral symbionts. Research works were started with sampling, bacterialisolation, anti-bacterial screening and
polyphasic identification. Results showed that 48 of 130 isolates were able to inhibit the growth of diseased isolates. Two isolates
of GSB18 and SMPSB4 showed a wide range of antibacterial activity. The molecular identification by partial 16S rRNA gene
sequencing revealed that they were closely related to the general Pseudoalteromonas and Pseudomonas. The sequence results of
anti-bacterial isolates indicated that there were two major groups of Actinobacteria and Gammaproteobacteria division.

©©2015
2015The Authors. Published
A. Sabdono, by Elsevier
P.H. Sawonua, B.V.Kartika.
A.G.D. This is anJ.M.
openAmelia,
access article under the CC
O.K. Radjasa. BY-NC-ND
Published license B.V.
by Elsevier
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014.
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014
Keywords: antipathogen; coral disease; genetic diversity; Panjang Island; postulat Koch.

*Corresponding author. Tel.: +62 813 2633 1329; Fax.: +62 247 460 039
E-mail address: ocky_radjasa@undip.ac.id

1876-6196 © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014
doi:10.1016/j.proche.2015.03.004
16 Agus Sabdono et al. / Procedia Chemistry 14 (2015) 15 – 21

Nomenclature

h hour (1 h = 60 min) μL microliter

mL mililitre DNA deoxyribose nucleic acid
g gram PCR polymerase chain reaction
CaCO3 CaciumCarbonat 16S rRNA ribosom Ribose Nucleic Acid
min minute (1 min = 60 sec)

1. Introduction

Coral reef ecosystems are very important for both ecologically and economically as they protect our coastlines
from erosion, providing essential habitat related to global nutrient cycling and tourism industry1. However, the
ecological and social benefits conveyed by these reefs are seriously threatened. Coral reefs are experiencing a recent
period of severe decline due to emerging coral diseases. Several mysterious coral diseases have been reported
worldwide and they continue to increase dramatically in the number of syndromes, host species and geographic
ranges2. These diseases are now recognized as one of the major causes of reef degradation and coral extinction.
In the last 30 years, there has been approximately a 30 % loss of corals worldwide, largely due to emerging
diseases3. The number and distribution of tropical coral diseases appear to be on the rise, with many information
being reported from widely studied regions such as the Caribbean4, the Pacific5 and Great Barrier Reefs6.
However, very little is known about coral disease and status in Indonesia, with only four recent published accounts
of coral disease in Wakatobi National Park7, Kep. Seribu8,Nusa Tenggara Timur9and Karimunjawa10. The increasing
of coral diseases over the last few decades has damaged the entire coral reef ecosystems and caused reef
deterioration4,11. Reports of disease and disease–like syndromes in reefbuilding corals have increased significantly
in which aproximately 35 diverse coral diseases and syndromes are described worldwide12.
Causative agents have been reported for only a few coral diseases. These are bleaching of Oculina patagonica by
the bacterium Vibrio shiloi13 black–band disease by a microbial consortium14, sea fan disease by the fungus
Aspergillus sydowii15, white pox disease by the bacterium Serratia marcescens16, and the Caribbean coral white
plague type II by the bacterium Aurantimonas coralicida17. Those situation opened up scientists to the realization
that coral diseases are becoming more widespread and pronounced around the globe. Coral diseases have been well
documented in the Caribbean, Great Barrier Reefs and IndoPasific4, 7, but little is known about Indonesia coral
diseases despite the region encompassing over 14 % of reefs worldwide and > 50 % of the world’s coral species.
Research reported regarding to coral diseases was causative agent of Black band diseaselike affected coral
Acropora sp. in Karimunjawa, Myroides odoratimimus strain BBD1, Bacillus algicola strain BBD2 and Marine
Alcaligenaceae bacterium strain BBD318.
Several strategies have been attempted to stop spreading and progressing of coral diseases. However, those
approaches were none have been attempted with any success on a large spatial scale. Alternative approaches to
identify coral diseases are by etiology and causality. It has been suggested that diseases in corals are the reflection of
complex interaction between the causative pathogens, host and environment. Bacteria are known to be the main
component of coral holobionts with higher diversity and are believed to respond in the disease events19. Thus,
profiling the bacterial community associated with coral diseases will play important role for better understanding of
emerging coral diseases20.There is now an urgent need to make an action to tackle this important issue about coral
diseases. Two important aspects need to be addressed, namely the thorough understanding on the causative agents of
particular diseases and the search for anticoral diseases obtained from coral bacteria. In this paper, only the second
aspect is presented.
 Agus Sabdono et al. / Procedia Chemistry 14 (2015) 15 – 21 17

2. Material and methods

2.1. Sampling and isolation of bacteria associated with coral

Specimens of the hard corals were collected by scuba diving at depths of 1 m to 3 m of the Panjang Island, Java
Sea, in May 2013 (S 07o 34.745ˈ ; E 110o 37.764ˈ). Individual specimens were placed separately into plastic bags to
avoid contact with air and brought to the surface. The samples were kept individually in plastic bags containing
natural sea water until processing within a few hours after collection. Tissue samples were removed from the
skeleton with a sterilized scrapper, and the exposed surface tissues were removed with a sterile scalpel blade. The
resultant tissues were then tenfold serially, diluted, spread on ½ strong ZoBell 2216E marine medium gelatin and
incubated at room temperature for 48 h. On the basis of morphological features, colonies were randomly picked and
purified by making streak plates.

2.2. Screening of the isolates for anti–pathogenic activity using overlay method

The bacterial isolates associated with healthy corals were tested for anti-pathogenic effect by the agar overlay
method against selected bacterial pathogens. Each of diseased bacterial isolates was used for anti-pathogenic effect
study. The 24 h old healthy coral isolates were spotted on the half–strength ZoBell 2216E marine medium gelatin
and incubated at room temperature for 4 d. All diseased isolates were cultured in Zobell 2216E marine medium
gelatin and the 18 hold cultures were used for the experiments. About 25 μL of the test cultures were suspended in
25 mL of Zobell 2216E soft medium gelatin and were poured immediately over the colonies of the healthy coral
bacteria on the plates. The plates were incubated at room temperature for 48 h. Antibacterial activity was defined by
the formation of inhibition zones around the bacterial colony.

2.3. Rescreening of the isolates for antagonistic activity using disc–diffusion method

All bacterial strains selected from overlay test were rescreened for their activity using gelatin disc diffusion
method22. Filter paper disks, 8 mm in diameter (Toyobo, Co, Japan), were placed on the surface of plates that
previously inoculated with50 μL of each diseased isolate. Then, 25 μL of selected isolates were put on each filter
discs. The plates were then incubated at 28 °C for approximately 72 h. At the end of incubation period, the zones of
inhibition were measured. Zone measurements were recorded as the distance from the edge of the zone to the edge
of the disk.

2.4. Microscopic and biochemical characterizations

Selected bacterial strains that consistently active on gelatin difused method were grown in Zobell 2216E medium
and underwent further microscopic and biochemical evaluations. Photomicrograph was used to determine the
morphology of the isolates. While standard gram staining, motility and biochemical characterizations based on
Bergey’s Manual of Determinative Bacteriology23 were used to determine their biochemical properties. Even both
microscopic and biochemical characterization were recorded but were not presented in this paper.

2.5. DNA Extraction, PCR amplification and sequencing of 16S rRNA gene fragment

Genomic DNA of selected antipathogenic isolates for PCR analysis was obtained from cell materials taken from a
gelatin plate, suspended in sterile water (Sigma, Germany) and subjected to five cycles of freeze (-80 °C) and thaw
(95 °C) Primers [(forward primer 8–27: 5’–AGAGTTTGATCCTGGCTCAG-3’24 and reverse primer 1510–1492:
5–GGTTACCTTGTTACGACTT–3’25 were used to amplify 16S rDNA. The resulting 16S rDNA sequences
corresponding to the genotype were analyzed for homologies with sequences in the data base using BLAST
searching. CLUSTAL X was used for multiple alignment/pairwise the DNA sequence26. Phylogenetic analysis was
performed with the Phylogenetic Analysis Using Parsimony (PAUP Ver.4) software package27.
18 Agus Sabdono et al. / Procedia Chemistry 14 (2015) 15 – 21

3. Results and discussion

3.1. Isolation and screening of anti—pathogeniccoral bacteria

Initial screening of healthy coral bacterial isolates against diseased coralisolates indicated that out of
130 coral bacterial isolates, 23 isolates (17.69 %) had antimicrobial activity at least three to any
pathogenic bacterial tested. These bacterial strains selected from overlay test were rescreened for their
activity by using gelatin disc diffusion method. The result showed that 10 isolates were still consistently
inhibiting the growth of pathogenic tested (Table 1).
Table 1. Antibacterial tested of selected isolates

Inhibition zone (mm) on:

No. Coral isolate Overlay 1 2 3 4

1. FS1 +++ 1.77 1.04 0.35 -

2. FSB2 +++ 3.55 0.53 0.49 -
3. FSB25 +++ 11.4 - 0.20 -
4. GSB18 +++++ - 0.14 1.20 -
5. GSB19 +++ - 0.79 0.20 7.9
6. GSB2.28 +++ 7.86 0.95 0.14 -
7. PSB5 +++ - - 1.58 -
8. PSB6 +++ - - 1.60 -
9. SMPSB4 +++++ - 0.31 2.67 3.1
10. SMPSB17 ++++ 3.05 0.53 0.31 7.5

Note: 1. White plague; 2. Yellow band; 3. Pink line syndrome; 4. Yellow blotch

The decrease of anti–pathogenicactivity using gelatin disc–diffusion method due to penetration of antibacterial
coral bacteria into the medium surrounding the disk was too slow to adequately show their potency to inhibit the
growth of pathogenic bacteria. When overlay method was used, there should be no diffusion problems.

3.2. Phylogenetic study

In this regard the 16S rDNA sequencing assist resolve the exact taxonomic position of coral bacteria, and
provided more detail information on their phylogenetic position among their closest relatives (Table 2 and Fig.1).

Table 2.Overview of 16S rRNA gene sequences retrieved from anti-pathogenic coral bacteria

No. Isolate Length Closest species Similarity Accession. Group

(bp) (%) Number

1. FS1 1134 Vibrio azureus CD21 99.0 KC210817 Gammaproteobacteria

2. FSB2 1979 Vibrio natriegenCM3 99.0 EU660320 Gammaproteobacteria
3. FSB2.5 1443 Pseudoalteromonas ruthenicaS2756 97.0 FJ457197 Gammaproteobacteria
4. GSB18 1208 Pseudoalteromonas aerugonisa R20 99.0 KJ631606 Gammaproteobacteria
5. GSB19 1479 Kocuria flava S43 96.0 JX007971 Actinobacteria
6. GSB2.28 1440 Pseudoalteromonas piscisida J38bio 96.0 JN681804 Gammaproteobacteria
7. PSB5 1305 Pseudomonasaeruginosa ACZ02 96.0 KJ667740 Gammaproteobacteria
8. PSB6 1302 Halomonas cupidaNBRC 100992 98.0 AB681327 Gammaproteobacteria
9. SMPSB4 1394 Pseudomonasaeruginosa SG 98,0 KJ99575 Gammaproteobacteria
10. SMPSB17 1690 Pseudomonas stutzeri Dr12 99.0 KF555605 Gammaproteobacteria
 Agus Sabdono et al. / Procedia Chemistry 14 (2015) 15 – 21 19

Fig. 1. Phylogenetic tree based on the 16S ribosomal DNA sequence data showing the relationships of representative strains with the most
closely related bacteria identified in the GenBank database. M. thermoautotropica DSM1974 was used as out group.
Boldface type indicates sequences from the present study.

In this study, members of the order Pseudomonadales and Alteromonadales were dominant, followed by
Vibrionales. Previous studies indicated the presence of some of these genera, including Pseudoalteromonas sp.and
Pseudomonas spp.28. Sequence types closely related (96 % or greater sequencehomology) to Vibrio azureus CD2,
Vibrio natriegen CM3, Pseudoalteromonasruthenica S2756, Pseudoalteromon asaerugonisa R20, Kocuria flava
S43, Pseudoalteromonas piscisida J38bio, Pseudomonas aeruginosa ACZ02, Halomonas cupida NBRC 100992,
Pseudomonas aeruginosa SG and Pseudomonas stutzeri Dr12 were found in coral tissue (Table 2). The
gammaproteobacteria dominated anti–pathogenic symbiont withmembers of the orders Vibrionales and
Pseudomonadales.
BLAST analysis indicated that two sequence types were related to the bacterial isolate that causes vibriosis in
cultured shrimp Penaeusvannamei boone, four sequence types were related to the bacterial isolate from ocean
surface waters. The detailed phylogenetic analysis showed that one of these sequences (FS1 isolate) was strongly
20 Agus Sabdono et al. / Procedia Chemistry 14 (2015) 15 – 21

supported (bootstrap value of 100 %) as a member of a clade including the Vibrionales group of
gammaproteobacteria (Fig. 1).
The present study indicates that using a culture dependent 16S rDNA–based approach it is possible to explore the
diversity of cultivable coral bacteria and their phenotypic traits. The implementation of the 16S rDNA approach has
developed the field of microbial ecology. The phylogenetic position of environmental bacterial populations in the
evolutionary tree could be determined and traced precisely by using this approach even in those complex
ecosystems. This approach will be useful to increase the knowledge on the physiological, biochemical, genetic, and
molecular properties of coral bacteria.

4. Conclusion

The finding of the preliminary data from this study showed that the microbial diversity in Panjang island was
high. Ten bacterias associated with corals were able to inhibit in vitro bacterial growth of bacteria isolated from
diseased corals. These bacterias could play a role in the microbial community dynamics of the corals limiting its
densities. Two isolates GSB18 and SMPSB4 showed a wide range of antibacterial activity. The molecular
identification by partial 16S rRNA gene sequencing revealed that they were closely related to the genera
Pseudoalteromonas and Pseudomonas. Gammaproteobacteria could be an important taxonomic group for future
studies due to a large number of this genus affected diseases. It is important to continue coral disease research by
expanding other parameters like season, time interval, and phytoplankton since this is likely to have a significant
impact on the types of culturable bacteria obtained.

Acknowledgements

Author acknowledge fieldwork provided by H. Nuryadi, Bayu and Tyas Akbar. Author are grateful for the
funding supported by grant from PNBP Diponegoro University under Graduated Team Research Grant scheme of
Diponegoro University (Hibah Penelitian Tim Pascasarjana, DIPA Undip No.: 023.04.2.189185/2014, Desember
2013).

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