Abstract: Background and Objective: Black Band Disease (BBD) causing tissue loss and mortality has come out as a serious threat to coral reefs. This disease has been reported from several coastal regions in the world, however little is known the procedures for controlling infectious diseases of this corals. The purposes of this study were to screen and characterize of coral-associated bacteria having antipathogenic potency against BBD coral disease. Materials and Methods: One hundred and eighty three healthy coral derived bacterial isolates were screened. Thirty four strains were selected for their ability to inhibit BBD pathogenic isolates. The disc-diffusion method were used to rescreen for their antipathogenic property against BBD pathogenic isolates. Then, the 16S rDNA approach and microbiological characteristics were used to identify bacterial selected. Results: Thirty four (18.58%) strains showed their ability to inhibit the growth of BBD pathogenic isolates. The NM1.2, NM1.8 and NM1.9 isolates were selected according to their consistency of BBD inhibition. The DNA sequences analyses showed these isolates belonged to only one major groups of γ-proteobacteria. The NM1.2, NM1.8 and NM1.9 isolates were all closely related to genus Pseudoalteromonas. Conclusion: Even though the low diversity of bacterial phylotypes were present within the coral family Acroporidae, however, this study provides the first evidence of acroporid coral bacteria possessing antipathogenic activity against BBD coral disease that can be recovered from corals. The sequence data of these antipathogenic strains, Pseudoalteromonas flavipulchra strain NM1.2, Pseudoalteromonas maricaloris strain NM1.8 and Pseudoalteromonas piscicida strain NM1.9, in this study have been deposited to the GenBank data library under the accession number LC184593, LC184594 and LC184595.
INTRODUCTION
Karimunjawa archipelago, which forms a chain of 27 islands, represents several ecosystem types such as coral reefs, mangroves and seagrass. This archipelago has been considered to be one of seven all Marine National Park in Indonesia since 2005 with more than 50 genera of corals1. Four among six extant hermatypic coral genera that include in Acroporidae family, Acropora sp., Montipora sp., Anacropora sp. and Astreopora sp., appear in Karimunjawa. This family exist in colonial, hermatypic that have all growth forms with small corallites and poorly developed columellae2. Acroporid coral was chosen as an object in the study because of the continues outbreak of BBD in this family that leads to degrading of coral ecology in Karimunjawa3.
During the last few years, Black Band Disease (BBD) has spread worldwide and caused large-scale mortalities and severe damage to coral reefs4-6. The BBD was first reported in 1973 in the Caribean7. Three decades after, BBD was first reported at Karimunjawa archipelago8 and has remained widespread since. This disease then has been observed throughout the Indonesia islands, affecting primarily the genera Montipora sp., Acropora sp., Diploastrea sp. and Pavona sp.9-11,3. The syndrome is characterized by a darkened areas, band-like microbial mat in the coral tissues at the interface between healthy coral tissue and exposed skeleton (Fig. 1). The BBD mat that composes the band is dominated by Phormidium corallyticum, genus Oscillatoria, sulfate-reducing bacteria Desulfovibrio spp. and sulfide oxidizing bacteria Beggiatoa spp6. However, recent studies utilized molecular techniques showed that newly bacterial strain Roseofilum reptotaenium presents in BBD mats12,13. The band moves across coral colonies that degrading, killing coral tissue, causing necrosis and colonizing by algae14,15. Progression rates of BBD lesions differ among host species, geographics and seasons. Sutherland et al.6 reported the progression rate of BBD in coral Carribean was 3 cm day–1, while Sato et al.16 reported the progression rate of BBD in 3.2-5.2 cm day–1. Four years after, Aeby et al.17 reported that the BBD pathogenic bacteria were more virulent with average rate of tissue loss of 5.7 cm2 day–1 over a 2 months period. Furthermore, Chen et al.18 and Sato et al.19 formulated the model to predict the dynamics of BBD development under environmental drivers from healthy coral colonies transitioning into a cyanobacterial patches and transitions into BBD. Garcia et al.20 reported coral metabolic status in health and disease that suffering from the increase in global and local threatening events.
The use of bacteria for the treatment of insect pest, weed and plant disease has had a long history21. Recently, antagonistic bacteria were used for biological control of pathogens infecting plant roots22. Numerous studies have demonstrated the ability of several antagonistic bacteria to suppress diseases caused by pathogens23. However, there are limited effort for preventing or treating infectious diseases of corals. Efrony et al.24 used bacterial viruses (bacteriophages) to control the spread of White Plaque (WP) coral diseases. While, Raymundo et al.25 attempted to stop the migration of BBD cyanobacterial mats from active lesions by covering the lesion with epoxy. However, none of them made any success on their efforts. The excellent approach is urgently needed to handle the spread of coral diseases and biological control might be the appropriate choice. The objectives of this study were to screen and identify antipathogenic BBD of bacteria associated with acroporid corals.
MATERIALS AND METHODS
Coral sampling and bacterial isolation: Acroporid corals, Acropora sp. and Montipora sp. are found on open reef bottoms of around Genting waters. Specimens of those hard corals were collected by scuba diving at depths of 5-6 m of the Genting island on March, 2015 (05̊50’44.3" LS 110̊35’0.07" BT).
| Fig. 1: | Black band disease on acroporid coral genus Montipora sp. |
| Fig. 2: | Sampling site locations |
Figure 2 shows the sampling site location. 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 seawater until processing within a few hours after collection. Bacterial isolation was conducted similar to previously study with slightly modification26. Coral fragments were rinsed with sterile seawater and scraped off with a sterile knife. The resultant tissues were serially diluted, spread on 1/2 strength ZoBell 2216E marine agar medium and incubated at room temperature for for 2 days. Based on of morphological characteristics, bacterial colonies were randomly selected and purified by streak method.
Antipathogenic assay: Acroporid bacterial symbionts were assayed for antipathogenic activity by overlay and agar diffusion methods as previously described27. Each BBD strains were designed for antipathogenic effect study. The ability of coral associated bacteria to inhibit the growth of BBD bacteria was performed by using an overlay test method. Aliquots culture of each BBD bacterium in the logarithmic growth phase (ca. 108 cells mL–1) was mixed with Zobell 2216E soft agar medium, which were then poured on to the respective agar surface, previously inoculated with coral associated bacteria and incubated for 96 h. The plates were then incubated at room temperature for 48 h. Antipathogenic activity was defined by the formation of inhibition zones around the bacterial colonies.
Nine coral bacteria selected from overlay test were reperformed by using the agar disc-diffusion method. Hundred microliters of each BBD isolate culture in the logarithmic phase (ca. 109 cells mL–1) was spread on agar medium. Paper disks (Ф 8 mm; Advantec, Toyo Roshi, Ltd., Japan) containing 35 μL of selected strain was placed on the respective agar surface. The plates were then incubated at room temperature for 48 h. Antipathogenic activity was defined by the formation of inhibition zones greater than 9 mm around the paper disk.
Microbiological characterizations: Coral selected bacteria 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 bacteriology were used to determine their biochemical properties.
Molecular taxonomy identification: The protocols of molecular identification were conducted according to previously a method described27. The DNA antipathogenic isolate for PCR analysis was gained from bacterial cells and extracted by using freeze and thaw method. Primers [(forward primer 8-27: 5’-AGAGTTTGATCCTGGCTCAG-3’28 and reverse primer 1510-1492: 5’-GGTTACCTTGTTACGACTT-3’29] 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 sequence30. Phylogenetic analysis was performed with the Phylogenetic Analysis Using Parsimony (PAUP version 4) software package31.
Nucleotide sequence accession numbers: Nucleotide sequences of the 16S rDNA from three antipathogenic BBD isolates obtained in this study have been deposited in the GenBank database under accession number LC184593, LC184594 and LC184595. The accession numbers of 16S rDNA of other strain cited and used as comparison.
RESULTS AND DISCUSSION
Recently, the number of coral diseases and the geographic distribution of diseases have reported increasingly. In concurrent, several studies have also reported the antimicrobial activity produced from scleractinian corals. Marquis et al.32 reported that several scleractinian species have antipathogenic activities. However, it is very few studies have been conducted to understand the role of coral associated bacteria in maintaining coral health33. In this study recorded that approximately 19% (34 isolates) healthy coral bacteria associated with acroporid corals demonstrated antipathogenic activity (Fig. 3). Similar result was reported by Ritchie34 that nearly 19% of the cultured bacteria isolated from the Acropora palmata showed antipathogenic activity against white pox coral disease. Shnit-Orland and Kushmaro35 showed that more than 20% (21) strains isolated from scleractinian corals demonstrated antipathogenic activity. While, Nissimov et al.36 found that only 9 (6%) strains isolated from healthy coral Oculina pathagonica showed antipathogenic activity against the causative agents Vibrio shiloi.
Based on the result of overlay tested, 9 isolates that capable of inhibiting more than 1 BBD isolate were re-examined by using agar disc-diffusion method to confirm the previous observation (Fig. 4). The experiment showed that NM1.8 and NM1.9, NM1.2 and TF1 isolates could inhibit the growth of 4, 4, 4 and 2 pathogenic BBD strains, respectively. While 5 other isolates could inhibit only 1 BBD isolate (Table 1). Inhibition zones that formed both on the overlay and the agar diffused methods indicated the possible production of cell associated antipathogenic BBD activity. This should be investigated further experiments to determine the nature of this interaction.
| Fig. 3: | Proportions of antipathogenic bacterial communities against the BBD, B0: No inhibition, B1: Inhibit 1 BBD, B2: Inhibit 2 BBD, B3: Inhibit 3 BBD, B4: Inhibit 4 BBD isolates |
| Fig. 4: | Antipathogenic assay on disc-diffusion agar method |
| Table 1: | Mean and Standard Deviation (Mean±SD) of antipathogenic assay by using diffusion agar |
| +: Inhibit 1 BBD isolate, ++: Inhibit 2 BBD isolates, +++: Inhibit 3 BBD isolates, ++++: Inhibit 4 BBD isolates | |
| Fig. 5: | 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 was used as out group |
| Table 2: | Characterization of representative BBD-antipathogenic coral bacteria |
It could be possible that coral mucus and its associated microorganisms play an important role in promoting beneficial microbial communities and the protection of the coral28,29.
The NM1.2, NM1.8 and NM1.9 isolates that could inhibit the growth of 4 pathogenic BBD strains were subjected to 16S rDNA sequence analysis. The sequencing results were analyzed to their homology to the sequences of known bacteria in the GenBank. The all sequences of the isolates have 98% similarity to the bacterial sequence in the GenBank (Table 2). All of these antipathogenic coral bacteria were belonged to γ-Proteobacteria group. Zhang et al.37 reported that the γ-Proteobacteria was the major bacterial group in scleractinian corals. In addition, Ibrahim38 reported that bacterial community of gorgonian coral Eunicea fusca were composed mainly of the bacterial group α-Proteobacteria and γ-Proteobacteria. The BLAST analysis showed that NM1.2, NM1.8 and NM1.9 strains were closely relative to Pseudoalteromonas flavipulchra strain NCIMB 2033, Pseudoalteromonas maricaloris strain KMM636 and Pseudoalteromonas piscicida strain NBRC 103038, respectively. In the previous study have shown similar genus Pseudoalteromonas to be present in healthy coral O. patagonica with antipathogenic property36. The 16S rRNA gene sequences of 3 bacterial selected isolates were subsequently aligned to construct a phylogenetic tree (Fig. 5). Molecular phylogenetic identification of strains revealed low diversity of cultivable bacteria which belonged to one genus only. Microbiological characterization demonstrated that NM1.2, NM1.8 and NM1.9 strains isolated from the tissues of acroporid corals were Gram-negative, aerobic bacteria and decomposed starch. The cells of these bacteria were rod-shaped with a single polar flagellum and they formed endospore (Table 3).
| Table 3: | Microbiological characterization of 3 antipathogenic BBD isolates |
| +: Reaction, -: No reaction, NC: No change, v: No assayed, F: Facultative | |
These characters were corresponds to the results of the molecular taxonomic identification. The comparison of taxonomic identification of these bacteria on their microbiological properties indicated that they were highly similar.
CONCLUSION
In conclusion, the diverse acroporid corals-derived bacteria from the Genting islands, Karimunjawa have a limited metabolites with various antipathogenic activities. The presence of γ-Proteobacteria may play significant roles in antipathogenic BBD communities. These bacterial group can be of potential use to biocontrol agents of BBD coral disease.
SIGNIFICANT STATEMENTS
| • | One hundred and eighty three bacteria of acroporid coral tissues was isolated, 19% of them could inhibit the growth of BBD pathogen |
| • | Three isolates identified as genus Pseudoalteromonas sp., presented consistent and strong antipathogenic BBD activity |
| • | These strains are useful as biocontrol agents, active against BBD coral pathogenic bacteria |
ACKNOWLEDGEMENT
The author is very grateful to Mr. Handung Nuryadi and Sakti Muchlisin as assistant researchers at Integrated Laboratory of Diponegoro Semarang. This research was supported by Penelitian Unggulan Perguruan Tinggi (PUPT): No. 022/SP2H/LT/ DRPM/II/2016 tanggal 17 Pebruari 2016.