Culture and Identification of Desulfovibrio Spp. from Corals Infected by Black Band Disease on Dominican and Florida Keys Reefs
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DISEASES OF AQUATIC ORGANISMS Vol. 69: 119–127, 2006 Published March 23 Dis Aquat Org Culture and identification of Desulfovibrio spp. from corals infected by black band disease on Dominican and Florida Keys reefs S. Viehman1, 3,*, D. K. Mills1, G. W. Meichel2, L. L. Richardson1 1Department of Biological Sciences and 2Southeast Environmental Research Center, Florida International University, University Park, Miami, Florida 33199, USA 3Present address: NOAA, National Ocean Service, Center for Coastal Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, North Carolina 28516, USA ABSTRACT: Black band disease (BBD) of corals is characterized as a pathogenic microbial consor- tium composed of a wide variety of microorganisms. Together, many of these microorganisms con- tribute to an active sulfur cycle that produces anoxia and high levels of sulfide adjacent to the coral surface, conditions that are lethal to coral tissue. Sulfate-reducing bacteria, as sulfide producers, are an important component of the sulfur cycle and the black band community. Previous molecular sur- vey studies have shown multiple Desulfovibrio species present in BBD but with limited consistency between bacterial species and infections. In this study we compared 16S rRNA gene sequences of sulfate-reducing bacteria selectively cultured from 6 BBD bands on 4 coral species, Diploria clivosa, D. strigosa, D. labyrinthiformes, and Siderastrea siderea, in the Florida Keys and Dominica. The 16S rRNA gene sequences were obtained through direct sequencing of PCR products or by cloning. A BLAST search revealed that 8 out of 10 cultures sequenced were highly homologous to Desulfovibrio sp. strain TBP-1, a strain originally isolated from marine sediment. Although the remaining 2 sequences were less homologous to Desulfovibrio sp. strain TBP-1, they did not match any other sulfate-reducing (or other) species in GenBank. KEY WORDS: Coral disease · Black band disease · Sulfate-reducing bacteria · Desulfovibrio · Florida Keys · Dominica Resale or republication not permitted without written consent of the publisher INTRODUCTION consortium (Richardson et al. 1997, Bythell et al. 2002, Richardson 2004). The microbial consortium is domi- Black band disease (BBD) of scleractinian corals is nated by cyanobacteria (Rützler & Santavy 1983, considered to be an important factor in the decline and Bythell et al. 2002, Cooney et al. 2002, Frias-Lopez et al. community restructuring of coral reefs (Rosenberg & 2003, Richardson 2004), sulfide-oxidizing bacteria pre- Loya 2004). This disease directly causes coral tissue sumed to be Beggiatoa spp. (Ducklow & Mitchell 1979, lysis as it progresses over an infected coral colony, lead- Viehman & Richardson 2002), marine fungi (Ramos- ing to either partial or complete death of the colony. Not Flores 1983), sulfate-reducing bacteria (SRB) including only is coral tissue destroyed by the disease, but it has Desulfovibrio (Chet & Mitchell 1975, Garrett & Duck- been shown that scleractinian coral recruitment onto low 1975, Cooney et al. 2002, Frias-Lopez et al. 2002), the newly available substrate of exposed coral skeleton and many other heterotrophic microbes (Cooney et al. produced by BBD is negligible (Edmunds 1991, Bruck- 2002, Frias-Lopez et al. 2002). Few members of the con- ner & Bruckner 1997, Kuta & Richardson 1997), further sortium have been cultured and no single or primary reducing coral cover on reefs (Kuta & Richardson 1996). pathogen has been identified. Characterized by a distinct black or dark red band, The complexity of the BBD microbial community has BBD is composed of a complex and distinct microbial recently been explored using molecular techniques, *Email: [email protected] © Inter-Research 2006 · www.int-res.com 120 Dis Aquat Org 69: 119–127, 2006 which revealed a high degree of microbial diversity strains were <97.5% similar in sequence homology to and variability among infections (Cooney et al. 2002, the closest related GenBank deposit, and only partial Frias-Lopez et al. 2002, 2003). However, the molecular gene sequences were used. Therefore, these results techniques used in these studies, while providing a are subject to some sequence bias and are probably broad survey of the BBD microbial community, may not indicative of a strict identification at the species not account for all bacteria present, particularly those level (Stackebrandt & Goebel 1994). Bacterial strains present at low densities. with >97.5% homology to Desulfovibrio salexigens BBD has an active sulfur cycle similar to that found in and Desulfovibrio sp. strain TBP-1 were found in one microbial mats in extreme environments such as hot- survey of partial bacterial sequences cloned from 3 dif- spring outflows and hypersaline ponds (Carlton & ferent BBD infections on different coral species in Richardson 1995). Both BBD and such microbial mats Curaçao (Frias-Lopez et al. 2002). No single Desul- contain anoxic and sulfide-rich microenvironments at fovibrio species was common to the different infec- depth in the mat. In the case of BBD, this zone is adja- tions. cent to the coral tissue, a condition which has been SRB were also found on denuded coral skeleton shown to cause coral tissue death (Richardson et al. behind BBD infections (Frias-Lopez et al. 2002). One 1997). In BBD, the source of this sulfide is biological, clone, sequenced from the bacterial community on the result of dissimilatory sulfate reduction (Richard- denuded coral skeleton, had 99% homology to Des- son 2004). SRB are ecologically significant in marine or ulfovibrio sp. strain TBP-1. Although multiple De- hypersaline microbial mat environments where, as the sulfovibrio species were found per sample from both dominant bacteria in the anaerobic zone, they may BBD and denuded coral skeleton behind the band, no oxidize up to 50% of total organic carbon in support single Desulfovibrio species was found on more than 1 of sulfate respiration (Howarth & Hobbie 1982, Jør- infection (Frias-Lopez et al. 2002). Additional clones gensen 1982). In seawater, the concentration of sulfate were reported as <97.5% homologous to D. zosterae is abundant at approximately 28 mM (Hansen 1993, and D. alaskensis, respectively. Clones identified Odom 1993), providing a sufficient sulfate supply for as most closely matching other genera of SRB were SRB to outcompete methanogens in an anaerobic also sequenced from denuded coral skeleton behind microenvironment, and thus act as terminal oxidizers the band, but again at <97.5% homology, thus not in the anaerobic degradation of organic matter to CO2 sufficient for a positive identification. These included (Jørgensen & Fenchel 1974). In a marine anoxic envi- Desulfofrigus oceanense strain Asv26, Desulfobotu- ronment, the influx of organic matter can be a control- lus sp. strain BG14, Desulfocella halophila, Desul- ling factor for the rate of sulfate reduction (Jørgensen forhopalus singaporensis, Desulfofrigus fragile, and 1982). Desulfobacterium catecholicum (Frias-Lopez et al. Desulfovibrio spp. are commonly found in marine, 2002). estuarine and freshwater environments that are both Perhaps the most obvious explanation for the varia- anaerobic and reducing (Widdel & Pfennig 1984). tion in Desulfovibrio species among BBD infections is Although SRB carry out a potentially integral role in that SRB fill an ecophysiological niche rather than a the etiology of BBD by producing toxic sulfide, charac- species-specific niche in BBD. Given the bias inherent terization of SRB associated with BBD has been limited in the techniques used, more Desulfovibrio species to date. SRB in BBD were first postulated to be Desul- may, in fact, have been present in the disease band fovibrio spp. in the 1970s (Garrett & Ducklow 1975). than were cloned or sequenced; thus only a subsample This was supported with fluorescence in situ of species present were detected in each sample. hybridization analyses using SRB probes and confocal While theoretically providing a broad survey of the laser microscopy of the BBD microbial community composition and variability of the BBD community, the (Schnell et al. 1996). Maximal growth of enrichment polymerase chain reaction (PCR) based approach cultures of SRB from BBD on hydrogen and lactate, employed by these studies has an inherent bias for when compared with other electron donors, supported under-representation of bacteria that may be present their identification as Desulfovibrio spp. (Schnell et al. at low densities. The question thus remains as to 1996). which, if any, microbial components of the disease are Two studies using culture-independent cloning of conserved between individual BBD infections and the 16S rRNA genes from BBD-associated microbial among various geographic areas. communities have shown a high diversity of bacteria The objective of this study was to culture and specif- within the disease consortium (Cooney et al. 2002, ically select for Desulfovibrio spp. from different BBD Frias-Lopez et al. 2002). These studies identified a infections on reefs in different regions of the variety of Desulfovibrio and other sulfate-reducing Caribbean. The 16S rRNA gene sequences were used genera present in BBD infections. Most of the cloned to compare the samples from this study to those found Viehman et al.: Desulfovibrio present in BBD of corals 121 in previous BBD studies as well as Table 1. Sampling location and host coral species for black band disease (BBD) to environmental SRB not associated sulfate-reducing bacteria culturing