The taxonomic position of the coral pathogen strain CBMAI 722T was determined on the basis of molecular and phenotypic data. We clearly show that the novel isolate CBMAI 722T is a member of the family Colwelliaceae, with Thalassomonas ganghwensis as the nearest neighbour (95 % 16S rRNA gene sequence similarity). CBMAI 722T can be differentiated from its nearest neighbour on the basis of phenotypic and chemotaxonomic features, including the utilization of cellobiose and l-arginine, the production of alginase and amylase, but not oxidase, and the presence of the fatty acids 12 : 0 3-OH and 14 : 0, but not 10 : 0 or 15 : 0. The DNA G+C content of CBMAI 722T is 39·3 mol%. We conclude that this strain represents a novel species for which we propose the name Thalassomonas loyana sp. nov., with the type strain CBMAI 722T (=LMG 22536T). This is the first report of the involvement of a member of the family Colwelliaceae in coral white plague-like disease

Barash, Y.

Rosenberg, E.

Sawabe, T.

Sharon, G.

Swings, J.

Thompson, F.L.

Open Marine Archive

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Thalassomonas loyana sp. nov., a causative agent
of the white plague-like disease of corals on the
Eilat coral reef
F. L. Thompson,1 Y. Barash,2 T. Sawabe,3 G. Sharon,2 J. Swings4
and E. Rosenberg2
Correspondence
F. L. Thompson
Fabiano.Thompson@terra.com.br
1Microbial Resources Division and Brazilian Collection of Environmental and Industrial
Micro-organisms (CBMAI), CPQBA, UNICAMP, CP 6171, Brazil
2Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life
Sciences, Tel Aviv University, Ramat Aviv 69978, Israel
3Laboratory of Microbiology, Research Faculty of Fisheries Sciences, Hokkaido University,
3-1-1 Minato-cho, Hakodate 041-8611, Japan
4Laboratory of Microbiology and BCCMTM/LMG Bacteria Collection, Ghent University,
K.L. Ledeganckstraat 35, Ghent 9000, Belgium
The taxonomic position of the coral pathogen strain CBMAI 722T was determined on the basis
of molecular and phenotypic data. We clearly show that the novel isolate CBMAI 722T is a member
of the family Colwelliaceae, with Thalassomonas ganghwensis as the nearest neighbour (95%
16S rRNA gene sequence similarity). CBMAI 722T can be differentiated from its nearest neighbour
on the basis of phenotypic and chemotaxonomic features, including the utilization of cellobiose
and L-arginine, the production of alginase and amylase, but not oxidase, and the presence of the fatty
acids 12 : 0 3-OH and 14 : 0, but not 10 : 0 or 15 : 0. The DNA G+C content of CBMAI 722T is
39?3 mol%. We conclude that this strain represents a novel species for which we propose the
name Thalassomonas loyana sp. nov., with the type strain CBMAI 722T (=LMG 22536T). This is
the first report of the involvement of a member of the familyColwelliaceae in coral white plague-like
disease.
There is growing concern for the health of coral reefs
worldwide (Rosenberg & Loya, 2004). Global climate
changes, sea water pollution from aquaculture, oil spills
and urban sewage, coral bleaching and other infectious
diseases have been deemed to be the main causes of the
decline of coral reefs (Hoegh-Guldberg, 2004; Hughes et al.,
2003; Knowlton & Rohwer, 2003; Rosenberg & Ben-Haim,
2002; Sutherland et al., 2004). Recent reports suggest that
bacteria may also play a role in the development of coral
tumours (Breitbart et al., 2005). Coral reefs may well serve as
indicators of both local environmental degradation and
global climate changes.
In the present study, we analysed the taxonomic position of
a novel isolate from the coral Favia favus suffering from
white plague in the Eilat coral reef (Barash et al., 2005). Pure
cultures of the strain caused the coral disease in controlled
aquarium experiments (Barash et al., 2005). 16S rRNA gene
sequence data positioned the novel isolate in the neighbour-
hood of the genus Thalassomonas (Macian et al., 2001). The
two currently recognized species of this genus, Thalasso-
monas viridans and Thalassomonas ganghwensis, have been
isolated from oysters in the Mediterranean sea and flat tide
sediments in Korea, respectively, but have not, to date,
ever been implicated in coral disease. Our data suggest that
strain CBMAI 722T represents a novel species of the genus
Thalassomonas.
Strain CBMAI 722T was isolated from diseased Favia favus
by crushing the coral in 10 ml sterile artificial sea water with
the aid of a mortar and pestle. The diseased Favia favus
was collected by SCUBA diving at Eilat in the Gulf of Aqaba,
Red Sea. Strain CBMAI 722T was grown on ZoBell 2216E
agar medium (MA; Oppenheimer & ZoBell, 1952) at 20 uC
for 48 h unless otherwise stated. Colony morphology was
examined on cultures grown on MA by using a stereoscopic
microscope. Cell morphology was examined on wet mounts
via a phase-contrastmicroscope. Exponentially growing cells
Published online ahead of print on 13 October 2005 as DOI 10.1099/
ijs.0.63800-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of Thalassomonas loyana sp. nov. CBMAI 722T is AY643537.
Additional phylogenetic trees and an electron micrograph of cells of
Thalassomonas loyana sp. nov. are available as supplementary figures in
IJSEM online.
63800 G 2006 IUMS Printed in Great Britain 365
International Journal of Systematic and Evolutionary Microbiology (2006), 56, 365–368 DOI 10.1099/ijs.0.63800-0
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in marine broth (MB) medium were negatively stained with
1% (w/v) uranyl acetate for electron microscopy (840A;
JEOL). Sequences for 16S rRNA genes were generated on a
DNA sequencer (ABI Prism 3100; Applied Biosystems) and
analysed as described by Thompson et al. (2001). The con-
sensus sequences were assembled and phylogenetic trees
were constructed based on the neighbour-joining (Saitou &
Nei, 1987), maximum-parsimony and maximum-likelihood
methods using KODON 2.0 software (Applied Maths). The
DNA G+C content was determined by HPLC (Tamaoka &
Komagata, 1984).
Phenotypic characterization of the isolate was performed
using API 20NE (bioMe´rieux) and Biolog GN2 metabolic
fingerprinting kits following the manufacturers’ instruc-
tions with some modifications. The bacterial inocula were
suspended in saline solution (3% NaCl) containing 10%
MB for Biolog tests, whereas cell suspensions (2% NaCl)
were used for API 20NE tests. Reactions were recorded
after 48 h at 30 uC. Carbon source utilization was also
checked with standard basal medium as described pre-
viously (Baumann et al., 1984; Leifson, 1963; Oppenheimer
& ZoBell, 1952). Salt tolerance was evaluated in a medium
consisting of 0?5 g yeast extract and 2?5 g peptone per litre
of sterile distilled water (Hidaka & Sakai, 1968). Alginate
hydrolysis activity was determined using previously des-
cribed methods (Sawabe et al., 1995). Antibiograms were
determined using the disc diffusion method of Acar &
Goldstein (1996) using commercial discs (Oxoid). Analysis
of fatty acid methyl esters was carried out as described by
Huys et al. (1994). For fatty acid analysis, cells were grown
on MA for 48 h at 28 uC.
According to our 16S rRNA gene sequence analysis, strain
CBMAI 722T is a member of the family Colwelliaceae
(Ivanova et al., 2004). The closest phylogenetic neighbours
of the novel isolate were T. ganghwensis KCTC 12041T and
T. viridans CECT 5083T, with 95 and 94% 16S rRNA gene
sequence similarity, respectively (Fig. 1). This low level of
similarity suggests that strain CBMAI 722T represents a
novel branch within the family Colwelliaceae. Strain CBMAI
722T consistently grouped with Thalassomonas species
even when different tree-building methods were used (see
Supplementary Fig. S1 in IJSEM Online).
Strain CBMAI 722T is a Gram-negative, motile, rod-shaped
bacterium with a single polar flagellum (see Supplementary
Fig. S2 in IJSEM Online). Many of the cells appear to divide
asymmetrically. The strain does not possess some of the
main characteristics of the genus Thalassomonas such as
oxidase activity and a DNA G+C value of between 42 and
48 mol%. Strain CBMAI 722T grew well in synthetic basal
sea water supplemented only with carbon sources and
without the addition of yeast extract, indicating that this
strain does not require organic factors for growth. Several
phenotypic and chemotaxonomic features were found that
can be used to discriminate strain CBMAI 722T from its
nearest phylogenetic neighbours (Table 1). For instance,
strain CBMAI 722T does not produce oxidase, does not
possess pigment and does not grow at 15 or 37 uC, all of
which are characteristics common to the other Thalasso-
monas species. It produces alginase, in contrast with the
other Thalassomonas species. Strain CBMAI 722T is able to
utilize cellobiose and L-arginine, but T. ganghwensis is not.
Previously recognized Thalassomonas species contain the
fatty acid 15 : 0, but this fatty acid is not found in strain
CBMAI 722T. In addition, strain CBMAI 722T has a higher
content of fatty acid 14 : 0 than the other Thalassomonas
species. The 16S rRNA sequence analysis appears to suggest
that strain CBMAI 722T may represent a new genus, but this
remains to be determined in future studies with a broader
collection of isolates of this novel taxon. We conclude that
strain CBMAI 722T represents a novel species for which we
propose the name Thalassomonas loyana sp. nov.
Description of Thalassomonas loyana sp. nov.
Thalassomonas loyana (loy9an.a. N.L. fem. adj. loyana
named in honour of the Israeli biologist Y. Loya).
Cells are 0?5–0?8 mm in width and 1–2 mm in length. They
form translucent, convex, smooth-rounded colonies with an
entire margin that are cream-coloured and 3 mm in size on
MA after 3 days incubation at 25 uC. No growth occurs on 0
or ¢ 10?0% NaCl. No growth occurs below 15 uC or at
37 uC or above. Tests negative for indole, arginine dihyd-
rolase, oxidase and urease activities. Weakly positive for
gelatinase activity and nitrate reduction. Does not ferment
glucose, but hydrolyses aesculin. According to Biolog tests,
Fig. 1. Phylogenetic tree with the estimated
position of Thalassomonas loyana sp. nov.
using the neighbour-joining method with the
Kimura two-parameter model based on the
almost-complete 16S rRNA gene sequences
with 500 bootstrap replications. Bar, 2%
estimated sequence divergence.
366 International Journal of Systematic and Evolutionary Microbiology 56
F. L. Thompson and others
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is able to utilize a-cyclodextrin, dextrin, glycogen, N-acetyl-
D-glucosamine, D-arabitol, D-cellobiose, i-erythritol,
D-galactose, a-D-glucose, maltose, D-raffinose, turanose,
acetate, citrate, D-gluconic acid, D-glucosaminic acid,
a-ketoglutaric acid, DL-lactate, malonate, propionate,
succinate, L-alanylglycine, L-glutamate, glycyl-L-aspartate,
glycyl-L-glutamate, L-histidine, L-proline, DL-carnitine, a-D-
glucose 1-phosphate and D-glucose 6-phosphate as sole
carbon sources. Does not utilize Tween 40, Tween 80, L-
arabinose, L-fucose, gentiobiose, myo-inositol, a-D-lactose,
lactulose, D-mannitol, methyl b-D-glucoside, D-psicose,
L-rhamnose, D-sorbitol, D-trehalose, xylitol, pyruvic acid
methyl ester, succinic acid monomethylester, cis-aconitic
acid, D-galactonic acid lactone, D-galacturonic acid,
D-glucuronic acid, a-hydroxybutyric acid, b-hydroxybutyric
acid, c-hydroxybutyric acid, p-hydroxyphenylacetic
acid, itaconic acid, a-ketobutyric acid, a-ketovaleric acid,
quinic acid, D-saccharic acid, sebacic acid, bromosuccinic
acid, succinamic acid, glucuronamide, L-alaninamide, L-
asparagine, L-aspartic acid, hydroxy-L-proline, L-leucine,
L-ornithine, L-phenylalanine, L-pyroglutamic acid, D-serine,
L-serine, L-threonine, c-aminobutyric acid, urocanic acid,
inosine, uridine, thymidine, phenyethylamine, putrescine,
2,3-butanediol, glycerol or DL-a-glycerol phosphate as
sole carbon source. Weakly positive for utilization of
N-acetyl-D-galactosamine, D-fructose, D-mannose, D-
melibiose, sucrose, formic acid, D-alanine, L-alanine and
2-aminoethanol according to the Biolog system. The major
fatty acids are summed feature 3 (31?3%; comprising
16 : 1v7c and/or 15 iso 2-OH), 14 : 0 (13?1%), 17 : 1v8c
(11?7%), 12 : 0 3-OH (6?3%), 18 : 1v7c (6?6%), 16 : 1v9c
(5?7%), 16 : 0 (4. 6%), 15 : 1v8c (3?1%), 11 : 0 3-OH
(2?5%), 12 : 0 (2?3%), 18 : 1v9c (1?9%), 13 : 0 (1?2%),
14 : 1v5c (1?2%) and 15 : 1v6c (1?2%). Produces amylase,
alginase, DNase and b-galactosidase, but not agarase or
k-carragenase. Sensitive to (mg per disc) erythromycin (10),
kanamycin (10), gentamicin (10), ampicillin (10) and
tetracycline (10). The DNA G+C content of the type strain
is 39?3 mol%.
The type strain of this species, CBMAI 722T (=LMG
22536T), was isolated from diseased coral in Eilat, Israel.
Acknowledgements
F. L. T. acknowledges a young researcher grant (2004/00814-9) from
FAPESP, Brazil. J. S. acknowledges grants from the Fund for Scientific
Research (FWO), Belgium. E. R. acknowledges support from the Israel
Center for the Study of Emerging Diseases.
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Table 1. Characteristics that differentiate Thalassomonas
loyana sp. nov. from other Thalassomonas species
Characteristics listed in this table were obtained from Macian et al.
(2001) and Yi et al. (2004). According to tests performed in
Baumann’s basal medium, T. loyana utilizes D-galactose, maltose,
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Characteristic T. loyana T. ganghwensis T. viridans
Pigment 2 Yellow Green
Production of:
Alginase + 2 2
Amylase + 2 +
Oxidase 2 + +
Utilization of:
Acetate + + 2
L-Arginine + 2 +
D-Cellobiose + 2 +
D-Galactose + + 2
D-Ribose 2 2 +
L-Tyrosine 2 + 2
Fatty acid content (%):
12 : 0 3-OH 6 2 5–6?1
14 : 0 13 2 2–3?1
10 : 0 2 4?9 2
15 : 0 2 1?2 6–11
16 : 0 5 22 11–13?7
16 : 1v9c 5?7 4?7 2
17 : 1v8c 12 4?4 14–19?7
16 : 1v7c and/or 15
iso 2-OH
31?3 20?6 21?2–28?4
Growth at 15 and 37 uC 2 + +
DNA G+C content
(mol%)
39?3 42 48?4
http://ijs.sgmjournals.org 367
Thalassomonas loyana sp. nov.
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368 International Journal of Systematic and Evolutionary Microbiology 56
F. L. Thompson and others


<i>Thalassomonas loyana</i> sp. nov., a causative agent of the white plague-like disease of corals on the Eilat coral reef

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