- Email: [email protected]
Coral Diseases in Panjang Island, Java Sea: Diversity of Anti–Pathogenic Bacterial Coral Symbionts
Available online at www.sciencedirect.com
ScienceDirect 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 Tropical Marine Biotechnology Research Center, Diponegoro University, Jl. Prof. H. Soedarto, Semarang 50275, Indonesia
b
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. © Authors. Published by Elsevier B.V.Kartika. This is anJ.M. openAmelia, access article under the CC BY-NC-ND license B.V. ©2015 2015The A. Sabdono, P.H. Sawonua, A.G.D. O.K. Radjasa. Published 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: [email protected]
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 mL g CaCO3 min
hour (1 h = 60 min) mililitre gram CaciumCarbonat minute (1 min = 60 sec)
μL DNA PCR 16S rRNA
microliter deoxyribose nucleic acid polymerase chain reaction ribosom Ribose Nucleic Acid
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
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.
17
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 No.
Coral isolate
Overlay
1
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
FS1 FSB2 FSB25 GSB18 GSB19 GSB2.28 PSB5 PSB6 SMPSB4 SMPSB17
+++ +++ +++ +++++ +++ +++ +++ +++ +++++ ++++
1.77 3.55 11.4 7.86 3.05
Inhibition zone (mm) on: 2 1.04 0.53 0.14 0.79 0.95 0.31 0.53
3
4
0.35 0.49 0.20 1.20 0.20 0.14 1.58 1.60 2.67 0.31
7.9 3.1 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
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
FS1 FSB2 FSB2.5 GSB18 GSB19 GSB2.28 PSB5 PSB6 SMPSB4 SMPSB17
Length (bp) 1134 1979 1443 1208 1479 1440 1305 1302 1394 1690
Closest species Vibrio azureus CD21 Vibrio natriegenCM3 Pseudoalteromonas ruthenicaS2756 Pseudoalteromonas aerugonisa R20 Kocuria flava S43 Pseudoalteromonas piscisida J38bio Pseudomonasaeruginosa ACZ02 Halomonas cupidaNBRC 100992 Pseudomonasaeruginosa SG Pseudomonas stutzeri Dr12
Similarity (%) 99.0 99.0 97.0 99.0 96.0 96.0 96.0 98.0 98,0 99.0
Accession. Number KC210817 EU660320 FJ457197 KJ631606 JX007971 JN681804 KJ667740 AB681327 KJ99575 KF555605
Group Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria Actinobacteria Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria
Agus Sabdono et al. / Procedia Chemistry 14 (2015) 15 – 21
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
19
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).
References 1. 2. 3. 4. 5. 6.
Voss JD. Coral reef & molecular ecology [Internet] accesed on October 12th 2014 from http http://www.fau.edu/hboi/meh/crme]. 2014. Voss JD, Mills DK, Remily ER, Myers JL, Richardson LL. Black band disease microbial community variation on corals in three regions of the Wider Caribbean. Microbiol Ecol.2007;54:730739. Harvell, D, Jordán-Dahlgren, Merkel S, et al.. Coral disease, environmental drivers, and the balance between coral and microbial associates. Oceanography 2007;20(B1):172195. Weil E, Smith G, Gil-Agudelo DL. Status and progressin coral reef disease research. Dis Aquat Organ 2006;69:1–7. Raymundo L, Harvell CD, Reynolds T. Poritesulcerative white spot disease: description, prevalence, and hostrange: A new disease impacting Indo-pacific reefs. Dis Aquat Organ 2003;56:95–104. Willis BL, Page CA, Dinsdale AD. Coral disease on the Great Barrier Reef. In: Rosenberg E, Loya Y, editors. Coral health and disease.
SpringerVerlag; 2004. Haapkyla J, Seymour, Trebilco AS. Smith D.Coral disease prevalence and coral health in the Wakatobi Marine Park, South–East Sulawesi Indonesia. J Mar Biol Assoc UK 2007;87:403–414. 8. Johan O, Bengen DG, Zamani NP, Suharsono. Distribution and abundant black band disease on corals Montiporasp in Seribu Islands, Jakarta. J of Ind Coral Reefs 2012:160–170. 9. Abrar M, Bachtiar I, Budiyanto A. Struktur komunitas dan penyakit pada karang (Scleractinia) di Perairan Lembata, Nusa Tenggara Timur [Community structure and scleractinian coral disease in Lembata waters, Nusa Tenggara Timur]. Indonesia J of Mar Sci 2012;17(2):109– 118.[Bahasa Indonesia]. 10. Sabdono A, Radjasa OK, Ambariyanto, et al. An early evaluation of coral disease prevalence on Panjang island, Java Sea, Indonesia. Int J Zool Res 2014;2:20–29. 11. Richardson LL, Goldberg WM, Carlton RG, Halas JC. Coral disease outbreak in the Florida Keys. Plague type II. Revis Biol Trop 1998;46:187–198. 12. Sussman M, Willis BL, Victor S, Bourne DG. Coral pathogens identified for white syndrome (WS) epizootics in the Indo-Pacific. PLoSONE 2008;3(6):e2393.
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Agus Sabdono et al. / Procedia Chemistry 14 (2015) 15 – 21 13. Kushmaro A, Rosenberg E, Fine M, Loya Y. Bleaching of the coral Oculinapatagonicaby Vibrio AK-1. Marine Ecology Progress Series 1997;147:159–165. 14. Carlton RG, Richardson LL. Oxygen and sulfide dynamics in a horizontally migrating cyanobacterial mat: black band disease of corals. FEMS MicrobiolEcol 1995;18:155–162. 15. Smith GW, Ives LD, Nagelkerken IA, Ritchie KB. Caribbean sea fan mortalities. Nature 1996;383:487. 16. Patterson KL, Porter JW, Ritchie KB,et al. The etiology of white pox, a lethal disease of the Caribbean Elkhorncoral, Acropora palmata. In: Cozzarelli NR, editor. Proceedings of the National Academy of Sciences of the United States of America 2002;99:8725–8730. 17. Aeby GS. Outbreak of coral diseas in the Northwestern Hawaiian Islands. Coral Reefs 2006;24:481. 18. Sabdono A, Radjasa OK. Karakterisasi molekuler bakteri yang berasosiasi dengan penyakit BBD (Black Band Disease) pada Karang Acropora sp. di Perairan Karimun Jawa. [Molecular characterization of bacteria associated with BBD (Black Band Disease) on coral Acropora sp. in Karimun Jawa waters]. Ilmu Kelautan 2006;11(3):158–162.[Bahasa Indonesia]. 19. Sunagawa S, Woodley CM, Medina M. Threatened corals provide underexplored microbial habitats. PLoS ONE 2010;5(3):e9554. 20. Work TM, Rameyer RA. Characterizing lesions in corals from American Samoa. Coral Reefs 2005;24:384–390. 21. Sutherland KP, Shaban S, Joyner JL, Porter JW, Lipp EK. Human pathogen shown to cause disease in the threatened eklhorn coral acroporapalmata. PLoS ONE 2011;6(8): 1-7. 22. Conception GP, Caraan GB, Lazaro JE Biological assays for screening of marine samples. In: Workbook second natural products workshop: Strategies in the quest for novel bioactive compounds from the sea. Marine Science Institute and Institute of Chemistry, University of Philippines. Deliman, Quezon City, Philippines; 1994. p.6–19. 23. Holt JG. Bergey's manual of determinative bacteriology. Baltimore: William & Wilkins; 1994. 24. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991;173:697–703. 25. Reysenbach AL, Giver LJ, Wickham GS, Pace NR. Differential amplification of rRNA genes by polymerase chain reaction. Appl Environ Microbiol 1992;58:3417–3418. 26. Thompson JD, Gibson TJ, Plewniak F, Jeanmougi F, Higgins DG. The Clustal X-Window interface: Flexible strategies for multiple alignment through sequence alignment aided by quality analysis tools. Nucl Acids Res 1997;25:4876–4882. 27. Sekar R, Longin T Kaczmarsky, Richardson LL. Effect of freezing on PCR amplification of 16S rRNA genes from microbes associated with black band disease of corals. Applied and environmental microbiology 2009;75(8):2581–2584. 28. Swofford DL. 1998. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. 143 pp.
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ScienceDirect 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 Tropical Marine Biotechnology Research Center, Diponegoro University, Jl. Prof. H. Soedarto, Semarang 50275, Indonesia
b
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. © Authors. Published by Elsevier B.V.Kartika. This is anJ.M. openAmelia, access article under the CC BY-NC-ND license B.V. ©2015 2015The A. Sabdono, P.H. Sawonua, A.G.D. O.K. Radjasa. Published 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: [email protected]
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 mL g CaCO3 min
hour (1 h = 60 min) mililitre gram CaciumCarbonat minute (1 min = 60 sec)
μL DNA PCR 16S rRNA
microliter deoxyribose nucleic acid polymerase chain reaction ribosom Ribose Nucleic Acid
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
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.
17
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 No.
Coral isolate
Overlay
1
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
FS1 FSB2 FSB25 GSB18 GSB19 GSB2.28 PSB5 PSB6 SMPSB4 SMPSB17
+++ +++ +++ +++++ +++ +++ +++ +++ +++++ ++++
1.77 3.55 11.4 7.86 3.05
Inhibition zone (mm) on: 2 1.04 0.53 0.14 0.79 0.95 0.31 0.53
3
4
0.35 0.49 0.20 1.20 0.20 0.14 1.58 1.60 2.67 0.31
7.9 3.1 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
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
FS1 FSB2 FSB2.5 GSB18 GSB19 GSB2.28 PSB5 PSB6 SMPSB4 SMPSB17
Length (bp) 1134 1979 1443 1208 1479 1440 1305 1302 1394 1690
Closest species Vibrio azureus CD21 Vibrio natriegenCM3 Pseudoalteromonas ruthenicaS2756 Pseudoalteromonas aerugonisa R20 Kocuria flava S43 Pseudoalteromonas piscisida J38bio Pseudomonasaeruginosa ACZ02 Halomonas cupidaNBRC 100992 Pseudomonasaeruginosa SG Pseudomonas stutzeri Dr12
Similarity (%) 99.0 99.0 97.0 99.0 96.0 96.0 96.0 98.0 98,0 99.0
Accession. Number KC210817 EU660320 FJ457197 KJ631606 JX007971 JN681804 KJ667740 AB681327 KJ99575 KF555605
Group Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria Actinobacteria Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria
Agus Sabdono et al. / Procedia Chemistry 14 (2015) 15 – 21
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
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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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