A disease outbreak on the coral Montipora aequituberculata from the Great Barrier Reef

Three replicate Montipora colonies displaying areas of apparently healthy coral tissue and black areas with obvious diseased lesions were sampled from a small reef flat area in Nelly Bay, Magnetic Island, Australia. 'Healthy' samples were collected from normally pigmented areas of a coral colony separated by &gt;5 cm from an observed lesion. 'Diseased' samples were collected from the boundary margin of healthy and diseased skeleton and tissue of black lesions. A permanent photographic monitoring site was established in the area in early January 2002.Samples were prepared in media designed for the isolation and enumeration of heterotrophic marine bacteria and Vibrio organisms respectively. Bacteria obtained from this study were designated: MA-HC1 and MA-HC2 for isolates obtained from healthy coral (HC) samples and MA-DC1 to MA-DC4 for isolates cultured from diseased coral (DC) samples, both grown on marine agar (MA) medium; and TCBS-DC1 to TCBS DC-4 for isolates cultured from diseased coral (DC) samples grown on TCBS growth medium.DNA was extracted from cultured isolates using the propriety reagent GeneReleaser and from coral samples using a modified UREA extraction buffer protocol. Universal bacterial primers 27F and 1492R, were used for amplification of 16S rRNA genes. PCR products were checked on a 1% agarose gel with bands of appropriate size either sequenced (bacterial isolates) or cloned (DNA extracted from corals). Duplicate PCR reactions were performed on DNA extracted from each coral sample and the products from the replicated reactions pooled.Amplified rDNA restriction analysis (ARDRA) was performed to analyse the diversity of clones within each of the healthy coral and disease coral libraries constructed.The diversity of clone libraries was further investigated by rarefaction analysis calculations. Other indices and models were used to analyse the variation of microbial diversity within clone libraries, with three different categories of measurement calculated with the EstimateS software package: Richness indices, Evenness indices, and Abundance models.Sequences of clones representing the OTU groupings generated were designated: HC-OTU1 to HC-OTU12 for clones from the healthy coral (HC) sample clone library, DC-OTU1 to DC-OTU12 for clones from the diseased coral (DC) sample clone library. Sequences were checked for chimera formation with the CHECK_CHIMERA software of the Ribosomal Database Project. Sequence data generated was analysed with the ARB software package. Tree topology was evaluated by reconstructing phylogenies using evolutionary distance (Jukes and Cantor model), maximum parsimony (ARB and DNAPARS) and maximum likelihood (ARB and fastDNAml) analyses of the aligned near full-length sequences extracted from the GenBank database.
 To report the results of culture-based andmolecular microbial analysis of both healthy and diseased coral tissue from Montipora corals at Magnetic Island undergoing atramentous necrosis and compare those data with 16S rDNA sequences from diseased lesions reported previously.
 The nucleotide sequence data appears in the GenBank database under the accession numbers: AY529868, AY529869, AY529870, AY529871, AY529872, AY529873, AY529874, AY529875, AY529876, AY529877, AY529878, AY529879, AY529880, AY529881, AY529882, AY529883, AY529884, AY529885, AY529886, AY529887, AY529888, AY529889, AY529890, AY529891, AY529892, AY529893, AY529894, AY529895, AY529896, AY529897, AY529898, AY529899, AY529900.

Organization: Australian Ocean Data Network