Genetic, morphological and growth characterisation of a new Roseofilum strain (Oscillatoriales, Cyanobacteria) associated with coral black band disease

Patrick Buerger, Carlos Alvarez-Roa, Karen D. Weynberg, Sebastien Baekelandt, Madeleine J.H. van Oppen

Black band disease (BBD) is a common disease of reef-building corals with a worldwide distribution that causes tissue loss at a rate of up to 3 cm/day. Critical for a mechanistic understanding of the disease's aetiology is the cultivation of its proposed pathogen, filamentous cyanobacteria (genus Roseofilum). Here, we optimise existing protocols for the isolation and cultivation of Roseofilum cyanobacteria using a new strain from the central Great Barrier Reef. We demonstrate that the isolation of this bacterium via inoculation onto agar plates was highly effective with a low percentage agar of 0.6% and that growth monitoring was most sensitive with fluorescence measurements of chlorophyll-a (440/685 nm). Cell growth curves in liquid and solid media were generated for the first time for this cyanobacterium and showed best growth rates for the previously untested L1-medium (growth rate k = 0.214 biomass/day; doubling time t gen = 4.67 days). Our results suggest that the trace metals contained in L1-medium maximise biomass increase over time for this cyanobacterium. Since the newly isolated Roseofilum strain is genetically closest to Pseudoscillatoria coralii, but in terms of pigmentation and cell size closer to Roseofilum reptotaenium, we formally merge the two species into a single taxon by providing an emended species description, Roseofilum reptotaenium (Rasoulouniriana) Casamatta emend. Following this optimized protocol is recommended for fast isolation and cultivation of Roseofilum cyanobacteria, for growth curve generation in strain comparisons and for maximisation of biomass in genetic studies.