NormFinder estimation of the optimum number of reference genes.

<p>Lowest accumulated standard deviation (Acc. SD) values indicate the optimal number of reference genes. The recommended upper limit for Acc. SD is set at 0.15.</p

Analysis of reference gene stability by GeNorm.

<p>Low stability values (M-values) indicate stable gene expression. Genes are ordered left to right in order of decreasing stability.</p

Descriptive statistics of reference gene expression by BestKeeper.

<p>Based on inspection of standard deviations (std dev; SD) genes can be ranked from the most stably expressed (lowest SD: <i>gyrA</i>), to the least stably expressed (highest SD: <i>gapB</i>). Genes with std dev [±CP]>1 are considered to be inconsistent. CP = crossing point or threshold cycle (Ct).</p

Summary of reference gene ranking by BestKeeper, NormFinder and GeNorm analysis.

<p>*Indicates the most stably expressed gene/s and + the least stably expressed gene as determined by each program.</p

Expression levels of reference gene candidates.

<p>A: Expression of reference genes across <i>A. brasilense</i> strains. B: Expression of reference genes across different culture conditions. Gene expression levels are represented by average Ct values. NB: nutrient broth; Flocc: Flocculation medium; N-free: Nitrogen free medium. Each bar represents the mean Ct values from three independent experiments. Error bars indicate standard error.</p

Specificity of qRT-PCR amplification.

<p>A: Melt curves for candidate reference genes. B: Agarose gel (2%) showing amplification of a single product of expected size for each candidate reference gene.</p

Phylogenetic tree showing the taxonomic relationships of culturable bacterial isolates.

<p><b>A.</b> All isolates. <b>B.. </b><i>Vibrio</i> spp. subtree <b>C.. </b><i>Rhodobactereacea</i> subtree. Phylogeny was inferred using the maximum likelihood method based on the Tamura-Nei model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044243#pone.0044243-Tamura3" target="_blank">[66]</a>. The tree displayed is the bootstrap consensus tree inferred from 500 replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The level of bootstrap support near the ends of some branches is low because these branches included several isolates with very high levels of sequence identity. All positions containing gaps and missing data were eliminated, leaving a total of 466 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044243#pone.0044243-Tamura1" target="_blank">[34]</a>. The evolutionary distances were computed using the Maximum Composite Likelihood method. The trees are drawn to scale, with the scale bars indicating the number of substitutions per nucleotide position. Clusters of organisms at branch ends share >99.5% sequence similarity, and are treated as the same organism for the purposes of this discussion. Markers on the left indicate the sample categories that each organism in the tree was isolated from (♦ exposed dead skeleton adjacent to living tissue of ASWS affected colonies (D), ▪ the interface between exposed skeleton and apparently healthy tissue at the margin of disease lesions (M), • the apparently healthy tissue adjacent to disease lesions on ASWS affected colonies (H), ▴ Apparently healthy colonies(C), ▾ related sequences retrieved from public databases).</p

Results of principal components analysis of the data generated by the culture-independent study.

<p>Results of principal components analysis of the data generated by the culture-independent study.</p

The Role of Coral-Associated Bacterial Communities in Australian Subtropical White Syndrome of <em>Turbinaria mesenterina</em>

<div><p>Australian Subtropical White Syndrome (ASWS) is an infectious, temperature dependent disease of the subtropical coral <em>Turbinaria mesenterina</em> involving a hitherto unknown transmissible causative agent. This report describes significant changes in the coral associated bacterial community as the disease progresses from the apparently healthy tissue of ASWS affected coral colonies, to areas of the colony affected by ASWS lesions, to the dead coral skeleton exposed by ASWS. In an effort to better understand the potential roles of bacteria in the formation of disease lesions, the effect of antibacterials on the rate of lesion progression was tested, and both culture based and culture independent techniques were used to investigate the bacterial communities associated with colonies of <em>T. mesenterina</em>. Culture-independent analysis was performed using the Oligonucleotide Fingerprinting of Ribosomal Genes (OFRG) technique, which allowed a library of 8094 cloned bacterial 16S ribosomal genes to be analysed. Interestingly, the bacterial communities associated with both healthy and disease affected corals were very diverse and ASWS associated communities were not characterized by a single dominant organism. Treatment with antibacterials had a significant effect on the rate of progress of disease lesions (p = 0.006), suggesting that bacteria may play direct roles as the causative agents of ASWS. A number of potential aetiological agents of ASWS were identified in both the culture-based and culture-independent studies. In the culture-independent study an Alphaproteobacterium closely related to <em>Roseovarius crassostreae</em>, the apparent aetiological agent of juvenile oyster disease, was found to be significantly associated with disease lesions. In the culture-based study <em>Vibrio harveyi</em> was consistently associated with ASWS affected coral colonies and was not isolated from any healthy colonies. The differing results of the culture based and culture-independent studies highlight the importance of using both approaches in the investigation of microbial communities.</p> </div

Comparative profiles of the bacterial communities associated with healthy and ASWS affected coral colonies.

<p>H - apparently healthy tissues of ASWS affected <i>T. mesenterina</i> colonies, M - the margin of disease lesions, D - exposed skeleton adjacent to the margin and C - healthy control colonies with no signs of ASWS. ‘OTU number’ refers to the number each OTU was assigned by the GCPAT clustering software. The bar at each position on the x-axis represents an OTU consisting of clones with identical hybridisation fingerprints. The height of the bars represents the mean number of clones per OTU for all replicate samples from each sample category. Only OTU’s containing ≥5 clones are shown.</p