Molecular detection and ecological significance of the cyanobacterial genera Geitlerinema and Leptolyngbya in black band disease of corals

Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus Geitlerinema and three to the genus Leptolyngbya. DGGE results, obtained using Cyanobacteria-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an Oscillatoria sp. The next most common sequence, 99% similar to that of the Geitlerinema BBD isolate, was present in three samples. One Leptolyngbya- and one Phormidium-related sequence were also found. Laboratory experiments using isolates of BBD Geitlerinema and Leptolyngbya revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of Geitlerinema in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium, Leptolyngbya.     

The presence of the cyanobacterial toxin microcystin in black band disease of corals

Black band disease (BBD) is a migrating, cyanobacterial dominated, sulfide-rich microbial mat that moves across coral colonies lysing coral tissue. While it is known that BBD sulfate-reducing bacteria contribute to BBD pathogenicity by production of sulfide, additional mechanisms of toxicity may be involved. Using HPLC/MS, the cyanotoxin microcystin was detected in 22 field samples of BBD collected from five coral species on nine reefs of the wider Caribbean (Florida Keys and Bahamas). Two cyanobacterial cultures isolated from BBD, Geitlerinema and Leptolyngbya sp. contained microcystin based on HPLC/MS, with toxic activity confirmed using the protein phosphatase inhibition assay. The gene mcyA from the microcystin synthesis complex was detected in two field samples and from both BBD cyanobacterial cultures. Microcystin was not detected in six BBD samples from a different area of the Caribbean (St Croix, USVI) and the Philippines, suggesting regional specificity for BBD microcystin. This is the first report of the presence of microcystin in a coral disease.     

Isolation and identification of eight microcystins from thirteen Oscillatoria agardhii strains and structure of a new microcystin.

Microcystins (cyclic heptapeptide hepatotoxins), isolated from 13 freshwater Oscillatoria agardhii strains from eight different Finnish lakes by high-performance liquid chromatography, were characterized by amino acid analysis, fast atom bombardment mass spectrometry (FABMS), and tandem FABMS (FABMS/collisionary-induced dissociation/MS). All strains produced two to five different microcystins. In total, eight different compounds, of which five were known microcystins, were isolated. The known compounds identified were [D-Asp3]MCYST (microcystin)-LR, [Dha7]MCYST-LR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, and [D-Asp3,Dha7]MCYST-RR. This is the first time that isolation of these toxins from Oscillatoria spp., with the exception of [D-Asp3]MCYST-RR, has been reported. Three of the strains produced a new microcystin, and the structure was assigned as [D-Asp3,Mser7]MCYST-RR. The structures of two new microcystins, produced as minor components by one Oscillatoria strain, could not be determined because of the small amounts isolated from the cells. Four strains produced [Dha7]MCYST-RR as the main toxin, but [D-Asp3]MCYST-RR was clearly the most abundant and most frequently occurring toxin among these isolates of O. agardhii.     

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain)

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.     

Adaptation of cyanobacteria to the sulfide-rich microenvironment of black band disease of coral

Black band disease (BBD) is a cyanobacteria-dominated microbial mat that migrates across living coral colonies lysing coral tissue and leaving behind exposed coral skeleton. The mat is sulfide-rich due to the presence of sulfate-reducing bacteria, integral members of the BBD microbial community, and the sulfide they produce is lethal to corals. The effect of sulfide, normally toxic to cyanobacteria, on the photosynthetic capabilities of five BBD cyanobacterial isolates of the genera Geitlerinema (3), Leptolyngbya (1), and Oscillatoria (1) and six non-BBD cyanobacteria of the genera Leptolyngbya (3), Pseudanabaena (2), and Phormidium (1) was examined. Photosynthetic experiments were performed by measuring the photoincorporation of [¹⁴C] NaHCO₃ under the following conditions: (1) aerobic (no sulfide), (2) anaerobic with 0.5 mM sulfide, and (3) anaerobic with 0.5 mM sulfide and 10 μM 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). All five BBD cyanobacterial isolates tolerated sulfide by conducting sulfide-resistant oxygenic photosynthesis. Five of the non-BBD cyanobacterial isolates did not tolerate sulfide, although one Pseudanabaena isolate continued to photosynthesize in the presence of sulfide at a considerably reduced rate. None of the isolates conducted anoxygenic photosynthesis with sulfide as an electron donor. This is the first report on the physiology of a culture of Oscillatoria sp. found globally in BBD.     

Cyanotoxins from Black Band Disease of Corals and from Other Coral Reef Environments

Many cyanobacteria produce cyanotoxins, which has been well documented from freshwater environments but not investigated to the same extent in marine environments. Cyanobacteria are an obligate component of the polymicrobial disease of corals known as black band disease (BBD). Cyanotoxins were previously shown to be present in field samples of BBD and in a limited number of BBD cyanobacterial cultures. These toxins were suggested as one of the mechanisms contributing to BBD-associated coral tissue lysis and death. In this work, we tested nine cyanobacterial isolates from BBD and additionally nine isolated from non-BBD marine sources for their ability to produce toxins. The presence of toxins was determined using cell extracts of laboratory grown cyanobacterial cultures using ELISA and the PP2A assay. Based on these tests, it was shown that cyanobacterial toxins belonging to the microcystin/nodularin group were produced by cyanobacteria originating from both BBD and non-BBD sources. Several environmental factors that can be encountered in the highly dynamic microenvironment of BBD were tested for their effect on both cyanobacterial growth yield and rate of toxin production using two of the BBD isolates of the genera Leptolyngbya and Geitlerinema. While toxin production was the highest under mixotrophic conditions (light and glucose) for the Leptolyngbya isolate, it was highest under photoautotrophic conditions for the Geitlerinema isolate. Our results show that toxin production among marine cyanobacteria is more widespread than previously documented, and we present data showing three marine cyanobacterial genera (Phormidium, Pseudanabaena, and Spirulina) are newly identified as cyanotoxin producers. We also show that cyanotoxin production by BBD cyanobacteria can be affected by environmental factors that are present in the microenvironment associated with this coral disease.     

Effects of light, temperature, nitrate, orthophosphate, and bacteria on growth of and hepatotoxin production by Oscillatoria agardhii strains.

The effects of bacteria, temperature, light, nitrate, and orthophosphate on growth of and hepatotoxin (desmethyl-3-microcystin-RR) production by Oscillatoria agardhii strains were studied under laboratory conditions. Strains were cultivated in Z8 medium under continuous illumination. Growth was determined by measuring dry weight and chlorophyll a, while toxin was analyzed by high-performance liquid chromatography. Two of the three toxic cultures studied produced more toxins in axenic than in nonaxenic cultures. High toxin production correlated with high nitrogen concentrations (test range, 0.42 to 84 mg of N per liter) and low light intensity (test range, 12 to 95 microeinsteins/m2 per s). Toxin production depended on phosphorus concentration at low levels of phosphorus (0.1 to 0.4 mg of P per liter) and higher concentrations had no additional effect. The optimum temperature for toxin production and growth of green O. agardhii was 25 degrees C. Red O. agardhii produced almost similar amounts of toxin at temperatures of 15 to 25 degrees C. The lowest toxin production by both strains was at 30 degrees C.     

Effects of light, temperature, nitrate, orthophosphate, and bacteria on growth of and hepatotoxin production by Oscillatoria agardhii strains

The effects of bacteria, temperature, light, nitrate, and orthophosphate on growth of and hepatotoxin (desmethyl-3-microcystin-RR) production by Oscillatoria agardhii strains were studied under laboratory conditions. Strains were cultivated in Z8 medium under continuous illumination. Growth was determined by measuring dry weight and chlorophyll a, while toxin was analyzed by high-performance liquid chromatography. Two of the three toxic cultures studied produced more toxins in axenic than in nonaxenic cultures. High toxin production correlated with high nitrogen concentrations (test range, 0.42 to 84 mg of N per liter) and low light intensity (test range, 12 to 95 microeinsteins/m2 per s). Toxin production depended on phosphorus concentration at low levels of phosphorus (0.1 to 0.4 mg of P per liter) and higher concentrations had no additional effect. The optimum temperature for toxin production and growth of green O. agardhii was 25 degrees C. Red O. agardhii produced almost similar amounts of toxin at temperatures of 15 to 25 degrees C. The lowest toxin production by both strains was at 30 degrees C.     

Adaptation to sulfide and to the underwater light field in three cyanobacterial isolates from Lake Arcas (Spain)

Abstract: Three strains of cyanobacteria isolated from karstic Lake Arcas were tested for photosynthetic adaptations to soluble sulfide. One of them, AO11, was identified as Oscillatoria cf. ornata , and forms dense populations in the sulfide-rich anoxic hypolimnion of this lake. This cyanobacterium was able to perform sulfide-dependent anoxygenic photosynthesis and its oxygenic photosynthesis was relatively insensitive to sulfide. The other strains studied were AP1 and AO21, identified respectively as Pseudanabaena sp. and Oscillatoria cf. tenuis , populations of which were present only in epilimnetic waters at low population densities. Pseudanabaena sp. also carried out anoxygenic photosynthesis, but oxygenic photosynthesis was totally inhibited by 0.5 mM sulfide. Oscillatoria cf. tenuis lost most of its oxygenic photosynthetic capacity when submitted to 0.1 mM sulfide and anoxygenic photosynthesis accounted for less than 20% of sulfide-free controls. In addition to different photosynthetic capabilities, the three cyanobacteria exhibited differences in light-harvesting photosynthetic accessory pigments. Pigment analysis of cultures grown under different light conditions showed the capacity of Oscillatoria cf. ornata AO11 to produce phycoerythrin under low light intensity or under predominantly green light, while neither Pseudanabaena sp. AP1 nor Oscillatoria cf. tenuis AO21 produced this pigment. The complementary chromatic adaptation of Oscillatoria cf. ornata correlates well with its summertime distribution under the dim light field of the hypolimnion. The distribution and abundance of specific cyanobacterial populations in Lake Arcas can thus be explained by the interplay of light regime and presence of sulfide as some of the most determinant ecological parameters.     

Distribution, host range and large-scale spatial variability in black band disease prevalence on the Great Barrier Reef, Australia

The prevalence and host range of black band disease (BBD) was determined from surveys of 19 reefs within the Great Barrier Reef Marine Park, Australia. Prevalence of BBD was compared among reefs distributed across large-scale cross-shelf and long-shelf gradients of terrestrial or anthropogenic influence. We found that BBD was widespread throughout the Great Barrier Reef (GBR) and was present on 73.7% of the 19 reefs surveyed in 3 latitudinal sectors and 3 cross-shelf positions in the summer of 2004. Although BBD occurred on all mid-shelf reefs and all but one outer-shelf reefs, overall prevalence was low, infecting on average 0.09% of sessile cnidarians and 0.1% of scleractinian corals surveyed. BBD affected approximately 7% of scleractinian taxa (25 of approximately 350 GBR hard coral species) and 1 soft coral family, although most cases of BBD were recorded on branching Acropora species. Prevalence of BBD did not correlate with distance from terrestrial influences, being highest on mid-shelf reefs and lowest on inshore reefs (absent from 66%, n = 6, of these reefs). BBD prevalence was consistently higher in all shelf positions in the northern (Cooktown/Lizard Island) sector, which is adjacent to relatively pristine catchments compared to the central (Townsville) sector, which is adjacent to a more developed catchment. BBD cases were clustered within reefs and transects, which was consistent with local dispersal of pathogens via currents, although the spread of BBD was not dependent on the density or cover of any of the coral taxa examined. In combination, these results suggest that BBD is part of the natural ecology of coral assemblages of the GBR, and its prevalence is relatively unaffected by terrestrial influences on the scales characteristic of cross-shelf gradients.     

Microcystins in natural blooms and laboratory cultured Microcystis aeruginosa from Laguna de Bay,Philippines

Laguna de Bay, the largest freshwater lake in the Philippines, experiences periodic blooms of the cyanobacteria Microcystis aeruginosa. Blooms of these cyanobacteria in 1996, 1998 and 1999 were sampled. HPLC and MALDI-TOF mass spectrometry were used to analyze for microcystins. A total of 16 structural variants of the toxin were isolated from the samples with microcystin LR (MC-LR) as the most abundant variant in the samples from 1996 and 1999 making up 77 to 85% of the total, respectively. MC-RR was the dominant variant in the 1998 bloom making up 38%. The samples from 1996 had the highest total toxin concentration (4049 microg g(-1)) followed by those from 1998 (1577 microg g(-1)) and 1999 (649 microg g(-1)). A strain of M. aeruginosa previously isolated from the lake was also cultured in the laboratory under different nitrogen concentrations (1, 3 and 6 mg L(-1)) and elevated phosphorus concentration (0.5 mg L(-1)) to determine the influence of these factors on toxin production. A total of 9 different structural variants of microcystin were isolated from the laboratory cultures with MC-LR consisting more than 75% of the total in all treatments. No significant differences in the total toxin concentration as well as the % distribution of the different variants among treatments were observed. However, the strain of M. aeruginosa cultured in the laboratory had from 3 to 20 times higher total microcystin than those harvested from the lake.     

Cloning of a DNA fragment encoding the 5'-terminus of the botulinum type E toxin gene from Clostridium butyricum strain BL6340

Chromosomal DNA was extracted from toxigenic Clostridium butyricum strain BL6340 isolated from a case of infant botulism. After digestion by EcoRI, a DNA fragment of about 1 kbp was cloned into Escherichia coli using lambda gt11, and was subcloned into pUC118. The E. coli cells transformed with this cloned fragment produced a 33 kDa protein which reacted with monoclonal antibodies recognizing the light chain (Lc) component of botulinum type E toxin. The nucleotide sequence of the cloned fragment was determined. The sequence was similar to that from botulinum type E toxin gene fragments previously determined by our laboratory (strains Mashike, Otaru and Iwanai). Several highly homologous sequences among the botulinum type A, C, E, butyricum and tetanus toxin genes were found in both translated and untranslated regions. These results suggest that the toxin gene of C. butyricum may have evolved by transfer from C. botulinum.     

Molecular Detection and Ecological Significance of the Cyanobacterial Genera Geitlerinema and Leptolyngbya in Black Band Disease of Corals

Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus Geitlerinema and three to the genus Leptolyngbya. DGGE results, obtained using Cyanobacteria-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an Oscillatoria sp. The next most common sequence, 99% similar to that of the Geitlerinema BBD isolate, was present in three samples. One Leptolyngbya- and one Phormidium-related sequence were also found. Laboratory experiments using isolates of BBD Geitlerinema and Leptolyngbya revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of Geitlerinema in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium, Leptolyngbya.     

A meta-analysis of 16S rRNA gene clone libraries from the polymicrobial black band disease of corals

Black band disease (BBD) is a polymicrobial disease affecting corals on reefs worldwide. Since 2002, researchers have constructed clone libraries from the BBD consortium using 16S rRNA gene primers targeting a variety of phyla. In the present study, a meta-analysis was conducted of published libraries from 2002 through the present that contain bacterial sequence data associated with individual clones and BBD samples. The libraries analyzed were from 87 BBD samples, collected from 16 species of scleractinian corals in 10 different geographic locations, and included 327 unique operational taxonomic units (OTUs). One OTU (cyanobacterial) was present in 62 (71%) samples, and three (one Cytophaga-Flavobacter-Bacteriodetes and two alphaproteobacterial) were present in 11 (13%) of the samples. The remaining 323 unique OTUs were present in <10% of the samples. The Alphaproteobacteria was the most diversely represented group. Analysis of clone libraries using nonmetric multidimensional scaling indicated strong regional specificity of BBD microbial populations, but limited host coral specificity. The results of this analysis support the hypotheses that: (1) a specific cyanobacterium may be the primary pathogen of BBD; (2) additional functional groups, required for BBD pathobiology, are represented by variable opportunistic species; and (3) opportunistic BBD microorganisms are primarily derived from the environment.     

微囊藻毒素降解菌的筛选、鉴定及其胞内粗酶液对藻毒素MCLR的降解

【目的】从巢湖底泥中分离筛选高效的藻毒素降解菌,并初步研究其胞内粗酶液降解藻毒素-LR(MC-LR)的特性,为水体中藻毒素污染的微生物治理提供有效的菌源与理论依据。【方法】利用富集驯化培养技术,以MC-LR为唯一碳源,分离筛选MC-LR降解菌,通过形态观察、生理生化实验及16S rRNA序列分析鉴定菌株,并考察其胞内粗酶液在不同条件下对MC-LR的降解特性。【结果】分离得到1株能高效降解MC-LR的菌株M6。分子鉴定结果表明,该菌株为蜡状芽胞杆菌(Bacillus cereus)。其降解MC-LR的活性物质为胞内酶,而且至少有3种酶参与了MC-LR的降解,它们是菌体本身的组织酶而非诱导酶。当反应体系pH值为8.0,胞内粗酶液浓度为404.9 mg/L,MC-LR的初始浓度为10 mg/L时降解率最高,16 h可达98.7%。【结论】分离出的MC-LR降解菌为蜡状芽胞杆菌,该菌株对MC-LR有较高的降解能力,并且酶促反应受到反应体系的pH值、胞内粗酶液浓度以及藻毒素初始浓度等因素的影响。     

The roles of temperature and light in black band disease (BBD) progression on corals of the genus Diploria in Bermuda

On Bermuda reefs the brain coral Diploria labyrinthiformis is rarely documented with black band disease (BBD), while BBD-affected colonies of Diploria strigosa are common. D. labyrinthiformis on these reefs may be more resistant to BBD or less affected by prevailing environmental conditions that potentially diminish host defenses. To determine whether light and/or temperature influence BBD differently on these two species, infection experiments were conducted under the following experimental treatments: (1) 26 °C, ambient light; (2) 30 °C, ambient light; (3) 30 °C, low light; and (4) 30 °C, high light. A digital photograph of the affected area of each coral was taken each day for 7 days and analyzed with ImageJ image processing software. The final affected area was not significantly different between species in any of the four treatments. BBD lesions were smaller on both species infected under ambient light at 26 °C versus 30 °C. Low light at 30 °C significantly reduced the lesion size on both species when compared to colonies infected at the same temperature under ambient light. Under high light at 30 °C, BBD lesions were larger on colonies of D. strigosa and smaller on colonies of D. labyrinthiformis when compared to colonies infected under ambient light at the same temperature. The responses of both species suggests that BBD progression on both D. strigosa and D. labyrinthiformis is similarly influenced by a combination of light and temperature and that other factors present before infections become established likely contribute to the difference in BBD prevalence in Bermuda.     

Black Band Disease Microbial Community Variation on Corals in Three Regions of the Wider Caribbean

Black band disease (BBD) is a pathogenic consortium of microorganisms that primarily affects massive framework-building scleractinian corals on reefs worldwide. There has been considerable debate concerning the microbial community composition of BBD. The aim of this study was to utilize microbial profiling to assess overall patterns of variation in the BBD bacterial community with respect to geographic location, host coral species, time, and nutrient regime. Length heterogeneity polymerase chain reaction (LH-PCR) was employed to differentiate BBD communities based on the natural variation in the sequence lengths within hypervariable domains of the 16S rRNA gene. Analysis of LH-PCR profiles of 97 BBD samples using multivariate ordination methods and analysis of similarity revealed significant clustering with respect to geographic region when comparing BBD sampled from reefs near Lee Stocking Island in the Bahamas’ Exuma Chain, the Northern Florida Keys (NFK), and St. John in the US Virgin Islands. There was much variability in BBD community composition on a regional basis, between sites in the NFK, and in terms of coral host species. The observed differences among BBD microbial community profiles were driven primarily by variation in relative abundance of 313–316-bp amplicons, which correspond to cyanobacteria and α-proteobacteria. The results obtained in this study support previous reports of intrinsic variability and complexity of the BBD microbial community but also suggest that this variability has biogeographic patterns.     

A comparison of toxins isolated from the cyanobacteria Oscillatoria agardhii and Microcystis aeruginosa

1. A toxin isolated from a strain of Oscillatoria agardhii var. was compared to a peptide toxin isolated from Microcystis aeruginosa. 2. The Oscillatoria toxin possessed similar hepatotoxic properties on mice as the Microcystis toxin but had a higher LD50 than the latter; 320 micrograms/kg compared to 43 micrograms/kg (i.p. mouse), respectively. 3. Ultra-violet and infra-red spectra showed that the Oscillatoria toxin is a peptide which is not identical to the Microcystis toxin. 4. The spectra also indicated some structural similarities in these toxins.     

Vero cytotoxin production and presence of VT genes in Escherichia coli strains of animal origin

Vero cytotoxin (VT) production has been studied in 34 Escherichia coli strains isolated from animals with enteric diseases. The strains were tested by DNA hybridization with probes for VT1 and VT2 sequences and also in toxin neutralization experiments with specific antisera. Twenty bovine strains, belonging to nine different O serogroups, produced VT1 or VT2 but not both toxins. In contrast, all 14 porcine strains of four different O serogroups produced VT2 only. Six of these porcine strains, belonging to serogroups O138, O139 and O141, were isolated from cases of oedema disease. In general, the porcine isolates produced toxin at a lower level than the bovine strains.     

Cyanobacteria associated with coral black band disease in Caribbean and Indo-Pacific Reefs

For 30 years it has been assumed that a single species of cyanobacteria, Phormidium corallyticum, is the volumetrically dominant component of all cases of black band disease (BBD) in coral. Cyanobacterium-specific 16S rRNA gene primers and terminal restriction fragment length polymorphism analyses were used to determine the phylogenetic diversity of these BBD cyanobacteria on coral reefs in the Caribbean and Indo-Pacific Seas. These analyses indicate that the cyanobacteria that inhabit BBD bacterial mats collected from the Caribbean and Indo-Pacific Seas belong to at least three different taxa, despite the fact that the corals in each case exhibit similar signs and patterns of BBD mat development.