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1 Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel 69978
2 Laboratory for Microbiology and BCCMTM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
Correspondence
E. Rosenberg
eros{at}post.tau.ac.il
| ABSTRACT |
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7c. The name Vibrio coralliilyticus sp. nov. is proposed for the novel coral-pathogenic species. In addition to strain YB1T, which was isolated from the Indian Ocean, five additional strains of V. coralliilyticus have been isolated, three from diseased P. damicornis in the Red Sea, one from diseased oyster larvae (Kent, UK) and one from bivalve larvae (Brazil). The six V. coralliilyticus strains showed high genotypic and phenotypic similarities and all were pathogenic to P. damicornis. The closest phylogenetic neighbours to V. coralliilyticus are Vibrio tubiashii, Vibrio nereis and Vibrio shilonii.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of strains LMG 10953, LMG 21349 and YB1T are AJ316167, AJ440004 and AJ440005.
| INTRODUCTION |
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Recently, we isolated a novel temperature-dependent pathogen of the coral Pocillopora damicornis, tentatively named Vibrio sp. YB1. At water temperatures above 26 °C, a pure culture of this pathogenic strain caused rapid destruction of the coral tissue within 2 weeks (Ben-Haim & Rosenberg, 2002
). The present study was carried out in order to characterize Vibrio sp. YB1 and related strains using a polyphasic approach.
| METHODS |
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Rep-PCR fingerprinting using GTG5 and BOX primers was performed as described previously (Rademaker et al., 1998
). Briefly, PCRs consisted of 1 µl template DNA (50 ng µl-1), 5 µl 5x times; Gitschier buffer, 0·4 µl BSA (10 mg ml-1), 2·5 µl DMSO (10 mg ml-1), 1·25 µl of a dNTP mixture (100 mM of each), 1 µl GTG5 primer or 1 µl BOX primer (both 0·3 µg µl-1; Amersham Pharmacia Biotech) and 0·4 µl Taq DNA polymerase (5 U µl-1; Goldstar Red). The PCR products generated with the GTG5 primer were electrophoresed in a 1·5 % agarose gel (w/v) and 1x times; TAE buffer at a constant 55 V for 900 min at 4 °C. The PCR products generated with the BOX primer were electrophoresed in a 2 % agarose gel (w/v) and 1x times; TBE buffer at a constant 130 V for 222 min at 4 °C. After staining with ethidium bromide, the digitized patterns were normalized and analysed numerically using the software BioNumerics 2.0. Similarity among patterns was calculated based on Pearson's similarity coefficient and dendrograms were built using UPGMA (Sneath & Sokal, 1973
).
16S rDNA sequencing.
Almost-complete 16S rDNA sequences were obtained following the methodology described previously (Thompson et al., 2001
). Briefly, fragments of the 16S rDNA were amplified by PCR using the conserved primers pA (16F27) and pH (16R1522) or MH1 (16F27) and MH2 (16R1485). Subsequently, purified products were used as templates for sequencing amplification using the ABI Prism BigDye Terminator Ready Reaction mix and eight primers (16F358, 16F536, 16F926, 16F1112, 16F1241, 16R339, 16R519 and 16R1093). Purified sequencing products were run on 48 cm denaturing polyacrylamide gels on an ABI Prism 377 DNA sequencer. Sequences were assembled with the AutoAssembler software (Applied Biosystems). The consensus sequences were transferred into BioNumerics 2.0 software, where a phylogenetic tree was constructed based on the neighbour-joining method (Saitou & Nei, 1987
). The phylogenetic positions of the consensus sequences were obtained using the program FASTA (Pearson & Lipman, 1988
) and compared to known Vibrio 16S rDNA sequences (Dorsch et al., 1992
; Ruimy et al., 1994
; Mellado et al., 1996
; Denner et al., 2002
).
DNADNA hybridization experiments and G+C content determination.
DNADNA hybridization was performed under stringent conditions using the microplate technique (Ezaki et al., 1989
) with photobiotin-labelled DNA at 39 °C for 3 h as described previously (Willems et al., 2001
). Hybridization values are means of the reciprocal and non-reciprocal values, each of which was performed in four replicates. The G+C content of DNA was determined by HPLC (Tamaoka & Komagata, 1984
).
Phenotypic characterization.
Colony morphology was examined using a stereoscopic microscope. Cell morphology was examined by scanning electron microscopy (JEOL 840A). Exponentially growing bacteria in MBT medium were adhered to a carbon-coated grid and negatively stained with 1 % uranyl acetate. Classical phenotypic tests were performed by standard methods (Farmer & Hickman-Brenner, 1992
). Biochemical tests were performed using the API 20NE system (micromethod tests for the identification of non-enteric Gram-negative rods; bioMérieux). The standard API 20NE protocol was used except that media were adjusted to 3 % NaCl. NaCl tolerance was determined in MBT medium containing varying concentrations (115 %) of NaCl. Sensitivity to the vibriostatic compound O/129 (2,4-diamino-6,7-diisopropylpteridine; Sigma) was determined after incubation for 48 h at 30 °C on MB agar containing 30 µg of the compound on a disc.
Sensitivity to antibiotics was examined using either the minimal inhibition concentration (MIC) method, for erythromycin (24 µg ml-1), tetracycline (20 µg ml-1), chloramphenicol (6 µg ml-1) and kanamycin (50 µg ml-1), or by the paper disc method, for penicillin (10 µg disc-1) and ampicillin (10 µg disc-1). Growth was also tested on MB agar containing 200 µg gentamicin ml-1.
Carbon utilization tests were carried out using Biolog GN2 MicroPlates. Pure cultures (1218 h growth) were harvested from MB agar plates and suspended in 20 ml GN/GP inoculating fluid (Biolog), adjusted to 3 % NaCl, to an OD600 of 0·1300·143. The suspension was then distributed into Biolog GN2 microwell plates. Each plate contained 96 microwells with one of 95 different carbon sources in each and tetrazolium violet as an indicator of metabolic activity. The plates were incubated for 48 h at 30 °C. Wells that changed to purple were marked as positive for metabolic utilization. Fatty acid analysis was performed following the protocol of the Microbial Identification System (Microbial ID Inc.) as described previously (Huys et al., 1994
). Strains for analysis were grown on TSA for 48 h at 28 °C.
The ability of each strain to infect the coral P. damicornis in controlled aquarium experiments was examined as described previously (Ben-Haim & Rosenberg, 2002
).
| RESULTS |
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-Proteobacteria using the FASTA program. These Vibrio strains formed a tight cluster, with more than 99 % 16S rDNA similarity (Fig. 2
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-D-glucoside,
-ketobutyric acid and alaninamide, whereas V. tubiashii and V. nereis do not. In contrast to the novel species, V. shilonii is negative for utilization of D-gluconic acid, L-leucine and D-serine. Moreover, the novel species possesses larger amounts of the fatty acids 17 : 0 and 17 : 0 iso than do V. tubiashii and V. nereis and larger amounts of 17 : 0 and 18 : 1
7c than does V. shilonii.
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| DISCUSSION |
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One of the most interesting findings of this study was the demonstration of the power of bacterial taxonomy to uncover novel coral-pathogenic strains. Vibrio strains LMG 10953 and LMG 20538, respectively isolated from diseased larvae of Crassostrea gigas and Nodipecten nodosus, were shown to be pathogenic to the coral P. damicornis. Strain LMG 10953 was previously identified as V. tubiashii (Hada et al., 1984
). Our results clearly demonstrate that this strain belongs to V. coralliilyticus sp. nov.
The fact that coral-pathogenic strains of V. coralliilyticus are widely distributed is important in understanding the source of coral disease and attempts to prevent their spread. V. coralliilyticus sp. nov. infects and causes tissue damage to its host coral only at water temperatures above 25 °C. It is likely that during the winter, when temperatures are lower, the bacterium is present in different hosts, possibly bivalve larvae. Clearly, the taxonomic studies described here open new and unexpected avenues of investigation regarding the transmission of coral diseases.
Description of Vibrio coralliilyticus sp. nov.
Vibrio coralliilyticus (co.ral.li.i.ly'ti.cus. L. n. corallium coral; Gr. adj. lytikos dissolving; N.L. adj. coralliilyticus coral-dissolving).
Conforms to the descriptions of the family Vibrionaceae and of the genus Vibrio (Farmer & Hickman-Brenner, 1992
). Cells are Gram-negative, non-spore-forming rods (1·21·5x0·8 µm) that are motile by a single polar, sheathed flagellum when grown on solid or liquid medium. Colonies are cream-coloured on marine agar and yellow on TCBS agar after 48 h incubation at 30 °C, have smooth edges and do not luminesce. The predominant cellular fatty acids are 16 : 0 and 18 : 1
7c. Biochemical tests and carbon compound utilization data are summarized in Table 4
. The type strain, strain YB1T (=LMG 20984T =ATCC BAA-450T), was isolated from a diseased coral of the species Pocillopora damicornis in the Indian Ocean. Further strains have been isolated from diseased P. damicornis in the Red Sea and from larvae of oysters in the Atlantic Ocean. All six strains described here are pathogenic to the coral P. damicornis. The G+C content of the type strain is 45·6 mol%.
| ACKNOWLEDGEMENTS |
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