INTRODUCTION

Coral reefs are widely known as some of the most productive ecosystems on earth and are certainly the most productive and species-rich environments in the oceans. Coral reefs provide critical protection to coastlines from storm damage, erosion and flooding by reducing wave action as well as the reservoir of marine natural products (Radjasa et al., 2011; Puspasari et al., 2011).

In addition to threats from human activities (Bryant et al., 1998), the emerging infectious diseases have now been regarded as serious threat for coral reefs around the world (Harvell et al., 2007). This emergence is believed has been fostered by ecological factors (Daszak et al., 2001) such as climate change (Rosenberg et al., 2007). The global warming issue in which an average of global temperature increased 0.6±0.2°C in the last century (IPCC, 2007), would definitely threaten coral reef ecosystems as it has been confirmed that certain coral diseases are indeed the result of the expression of temperature-regulated bacterial virulence genes (Rosenberg and Ben-Haim, 2002).

White band disease is among the reported coral diseases that commonly found in branching corals such as Acropora spp. (Smith and Thomas, 2008). It has been suggested that, diseases in corals are the reflection of complex interaction between the causative pathogens, hosts and environment. Bacteria are known to be the main component of coral holobionts with higher diversity and are believed to respond in the disease events (Sunagawa et al., 2009). Thus, profiling the bacterial community associated with coral diseases will play important role for better understanding the emerging coral diseases (Work et al., 2008).

Karimunjawa islands, in the North Java Sea are part of marine national park. Incidences of white band disease among branching corals Acropora spp. have been observed, however, less reports have documented on the diversity of microbial community associated with the healthy and diaseased Acropora from this area. Here, we report the diversity of the causative agents of white band disease of bacteria associated corals based on culture-dependent approach.

MATERIALS AND METHODS

Sampling site: Diseased coral Acropora species were determined by using Manta tow technique. Upon the finding of diseased and healthy colonies at a depth of approximately 3 meters, mucus samples were collected in situ from the surfaces of 3 healthy and 3 diseased corals using a 20 mL syringe by scuba diving from Tanjung Gelam waters, Karimunjawa islands, North Java Sea (Fig. 1). Upon collection, syringes were put onto strerile Whirlpak bags. Upon retrieval, all sample bags were put on coolbox until further preparation.

Bacterial isolation: Coral mucus from 3 healthy and 3 diseased corals Acropora humilis and Acropora tortuasa were serially diluted with sterile seawaters and spread onto each respective surface agar of ZoBell 2216E medium and incubated for 48 h. The appeared colonies were purified and isolated by using streak method.

Fig. 1:	Sampling site for the collection of coral Acropora spp. from Tanjung Gelam waters

DNA extraction, amplification and rapid grouping: Genomic DNA of each bacterial isolate from both healthy and diseased corals were obtained by freeze and thaw (Radjasa et al., 2007a). A total of 19 isolates were grouped based on rep-PCR as previously reported methods (Radjasa et al., 2007b; Sarjito et al., 2009).

Antagonistic test: Ten representative isolates from both healthy and diseased corals based on constructed dendrogram of rep-PCR were tested for antagonistic against each other. One petri dish containing medium ZoBell 2216E was inoculated with a 24 h old of each difffent isolate grown in a ZoBell 2216 broth medium and spread with glass spreader. Nine sterile paper discs were put onto the agar surface which were then poured with a 20 μL of each of the remining isolates. The petries were incubated for 24 h and were checked for the presence of inhibition zones in the surrounding each paper disc.

Infection test: Ten isolates that all passed the antagonistic test were prepared to form a bacterial consortium by inoculating them onto one single flash containing 100 mL ZoBell 2216E broth medium. The flash was incubated for 2x24 h and was checked for density by using a spectrophotometer at 550 nm. Two months old colonies of Acropora tortousa and Acropora humilis were put onto controlled aquariums and were acclimated for 10 days. Both corals were then inoculated with a 5 mL of bacterial consortium by using a syringe and were observed daily for the occurrence of white band symptons.

DNA sequencing: PCR amplification of partial 16S rRNA genes of bacterial isolates, purification of PCR products and subsequent sequencing analysis were performed according to Radjasa et al. (2007a). The determined DNA sequences of strains were then compared for homology to the BLAST database. A phylogenetic tree was constructed using maximum-likelihood analysis and phylogenetic analysis was performed with the PAUP software package (Radjasa and Sabdono, 2008).

DNA sequences: DNA sequences of all bacterial isolates have been deposited in the DNA Database Bank of Japan (DDBJ) in the following accession numbers: AB675037-675045.

RESULTS

A total of 19 bacterial isolates were obtained from both healthy and diseased corals A. tortuosa and A. humilis (Table 1). All isolates were successfully grouped by using a rep-PCR technique (Fig. 2) followed by dendrogram analysis (Fig. 3) resulted in the occurrence of 10 different group representing bacterial isolates of both healthy and diaseased corals.

Based on the constructed phylogenetic tree, it was shown that 10 representative isolates were closely affiliated with the following bacterial genera of Vibrio, Pseudoalteromonas and Bacillus (Fig. 4).

The antagonistic test revealed that none of the 10 representative isolates based on rep-PCR analysis showing any antagonistic activity against each other. Therefore, it satified the requirement to form a bacterial consortium needed for infection test.

Following infection test on 2 months old corals A. humilis and A. tortuosa by a bacterial consortium, it was indeed observed that white band disease symptons occurred on the infected corals.

Table 1:	List of isolates collected from healthy and diseased corals

Fig. 2:	Gel electrophoresis of rep-PCR of bacterial isolates

Fig. 3:	Constucted dendrogram based on rep-PCR analysis

Fig. 4:	Phylogenetic tree of the causative agent of white band disease

It interesting to note that bacterial consortium caused a white band disease type I on coral A. humilis and white band type II on coral A. tortuosa after 4 and 11 days of incubation, respectively (Fig. 5).

Fig. 5(a-b):	Infection test by bacterial consortium on coral A. humilis (a) and A. tortuosa (b)

DISCUSSION

Water temperature, is among ecological factors that exhibited statistically significant relationships with coral diseases (Rosenberg et al., 2007). The emergence of coral diseases as a result from seawater temperature rise has become a serious threat for the sustainable use of coral reefs. Unfortunately, one of the yet least understood aspects on coral diseases is the diversity of the causative agents of particular disease. In addition, the understanding on the diversity of causative agents agents also plays important role in the management of coral diseases, especially to prevent the spreading of particular disease in particular location, season and ecological parameter.

Present results highlight the diversity of the causative agents of white band disease of culturable bacteria associated with healthy and diseased corals A. humilis and A. tortuosa. Despite the fact that there is a disparity between culture-dependent and culture-independent approaches (Pantos and Bythell, 2006), in estimating the bacterial community associated with corals, isolation of culturable parts of these associants would help in profiling the causative agents of white band disease in coral Acropora spp.

Rapid grouping based on rep-PCR techniques followed by the construction of dendogram allowed the estimation of richness of culturable bacterial community associated with healthy and diseased corals. This study supports the power of rep-PCR in determining the diversity of marine bacteria as previosly reported (Radjasa et al., 2007b; Sarjito et al., 2009).

It is interesting to note that 10 isolates representing the diversity of both healthy and diseased corals showed no antagonistic activity among each other. Thus this fact satified the requirement of forming a bacterial consortium needed for infection test.

The present study indicates that despite the same composition of bacterial consortium infected both corals, the white symptons produced by the consortium observed in two different coral species were indeed different. In the controlled aquariums, colony of coral A. humilis showed white band disease type I in which the infection started from the base of coral speading into the tip (Rosenberg et al., 2007). On the other hand, coral A. tortuosa showed white band disease type II (Ritchie and Smith, 1995) where, the infection started from the branch and moved forward the tip of branch. In addition, both corals showed different vulnerability toward the infection, where in A. humilis the infection was observed on the 4 days and in A. tortuosa was on the day 11 days after inoculation.

Molecular identification of the causative agents of white band disease revealed that the generic composition of these causative agents included the member of Vibrio, Pseudoalteromonas and Bacillus. There is no significant differences on the diversity of the causative agents from culturable bacterial community between healthy and diseased corals. This finding was in accordance with a previous results in which no dramatic differences in the bacterial community between healthy and white band disease based on culture-independent analysis (Casas et al., 2004). The present study shows that, the identification of bacteria in healthy corals that were similar to known pathogens or bacteria that were previously isolated from diseased, stressed or injured marine invertebrate (Sunagawa et al., 2009), may suggest a role of exogenous opportunistic pathogens of broad host range. Alternatively, colonization by opportunistic pathogens or uncontrolled growth of commensals may have taken advantage of a compromised host immune system caused by a primary agent and/or unfavorable environmental conditions.

The fact the bacterial consortium was the causative agent of white band disease in corals A. tortuosa and A. humilis in the present study, supports the previous argument (Richardson, 2004) that coral disease is not caused by single pathogen, but rather a pathogen consortium.

ACKNOWLEDGMENTS

This study was partially funded by a grant from The US Department of State’s Biosecurity Engagement Program (BEP), USA (grant # IDB1-21003-JA-08) through the US Civilian Research and Development Foundation (CRDF). Hasbullah Iwansyah Hakim was awarded graduate research grants from Agency for Education and Training, Ministry of Marine Affairs and Fisheries, Indonesia (Contract No. Pusdik KKP.B09/SJ.2/KP.510/III/2010) and COREMAP II (Coral Reef Rehabilitation and Management Program), Ministry of Marine Affairs and Fisheries, Indonesia (Contract.No. PMO/NCU COREMAP II. 24/KP3/2010).