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Vol. 75, No: 8, 2009, Pages:

Effect of Freezing on PCR Amplification of 16S rRNA Genes from Microbes Associated with Black Band Disease of Corals+

Raju Sekar,* Longin T. Kaczmarsky, and Laurie L. Richardson

Biological and Environmental Systems Group, Department of Chemical and Process Engineering, The University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom.


Molecular analysis of black band disease of corals revealed that samples frozen immediately after collection yielded more proteobacterial 16S rRNA sequences, while unfrozen samples produced more cyanobacterial and sulfur-oxidizing bacterial sequences. These results suggest the need to use multiple approaches for preparation of samples to characterize this complex polymicrobial disease. Black band disease (BBD) is a polymicrobial disease that affects corals on reefs worldwide. It consists of a migrating microbial mat dominated by cyanobacteria that lyses coral tissue, leading to coral colony death, and is one of the most destructive of coral diseases. Microscopic examination of BBD samples consistently reveals an abundance of nonheterocystous, filamentous cyanobacteria and colorless gliding bacteria with internal elemental sulfur granules characteristic of the genus Beggiatoa (6, 17, 18). It is thought that these are key players in the etiology of BBD. However, with one exception (2), previous molecular studies of BBD consistently detected very low proportions of cyanobacteria (4, 8, 9, 19, 20) and only one study has detected Beggiatoa (19). Instead, all molecular BBD studies indicate a highly variable and diverse composition of heterotrophic bacteria, mostly members of the Alphaproteobacteria.

Keywoards:Flavobacterium columnare, Escherichia coli,16S rRNA Genes,Beggiatoa filaments,Alphaproteobacteria,plant growth.

Corresponding author: Tel 44 114 222 0062, Fax 44 114 222 7501.

E-mail: S.Raju@Sheffield.ac.uk


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