Aims:  To develop and test a real‐time PCR assay to detect and quantify genes specific to Cylindrospermopsis sp. and cylindrospermopsin‐producing cyanobacteria. Method and Results:  A duplex real‐time PCR assay was developed that targets a cylindrospermopsin‐specific and Cylindrospermopsis raciborskii‐specific DNA sequence. The C. raciborskii‐specific sequence was based on the rpoC1 DNA‐dependent RNA polymerase gene, whilst the cylindrospermopsin‐specific sequence was selected by surveying an extensive number of potential cylindrospermopsin‐producing cyanobacterial strains for genes implicated in toxin production, aoaA, aoaB and aoaC. In toxic strains, sequences of each of these three genes were always present; whilst in nontoxic strains the distribution of these sequences was patchy, resulting in what are likely to be natural deletion mutants. The real‐time assay was optimized on a fixed and portable device, with results indicating that the reliable limit of detection for the assay was 100 copies per reaction or 1000 cells ml−1 for both target sequences on both devices. In routine environmental samples enumerated by microscopy, the assay results were positive for all samples where C. raciborskii cells were observed at >1000 cells ml−1 and negative in 15 samples where no C. raciborskii cells were observed. In field samples, the number of copies of the rpoC1 sequence more closely approximated the number of cells enumerated by microscopy, the number of copies of the pks sequence and detection of the toxin‐specific sequence matched the results of toxin testing. Conclusions:  The duplex real‐time PCR assay was a sensitive and rapid method for detecting potential cylindrospermopsin‐producing cyanobacteria in the laboratory or in the field. The observation of probable natural deletion mutants provides further evidence that the aoaA, aoaB and aoaC genes are involved in toxin production. Significance and Impact of the Study:  This assay provides a new monitoring capability for tracking cylindrospermopsin‐producing cyanobacteria that are an emerging threat to water quality.

The taxonomy and toxicity of a single-filament isolate from a filamentous cyanobacterial bloom in Lake Hakanoa (New Zealand) were examined by microscopy and liquid chromatography-mass spectrometry. Based on a morphological examination of environmental and cultured material, strain CAW-BG02 was identified as Raphidiopsis mediterranea Skuja; however, subsequent phylogenetic analysis of the 16S rRNA gene sequence demonstrated that CAWBG02 was most likely to be a single culture of Aphanizomenon issatschenkoi (Usacev) ProshkinaLavrenko. Toxin testing confirmed that the original bloom and A. issatschenkoi isolate produced anatoxina but did not produce homoanatoxin-a or any cylindrospermopsins, saxitoxins, or microcystins. Despite the absence of cylindrospermopsin production, genes implicated in the biosynthesis of cylindrospermopsin were successfully amplified from A. issatschenkoi strain CAWBG02. To our knowledge, this is the first confirmation of an anatoxin-a-producing species in the Southern Hemisphere and the first report of anatoxin-a production by A. issatschenkoi.

No abstract

Cyanobacterial composition of microbial mats from an alkaline thermal spring issuing at 43-71 degrees C from tropical north-eastern Australia are described using a polyphasic approach. Eight genera and 10 species from three cyanobacterial orders were identified based on morphological characters. These represented taxa previously known as thermophilic from other continents. Ultrastructural analysis of the tower mats revealed two filamentous morphotypes contributed the majority of the biomass. Both types had ultrastructural characteristics of the family Pseudanabaenaceae. DNA extracts were made from sections of the tentaculiform towers and the microbial community analysed by 16S cyanobacteria-specific PCR and denaturing-gradient gel electrophoresis. Five significant bands were identified and sequenced. Two bands clustered closely with Oscillatoria amphigranulata isolated from New Zealand hot springs; one unique phylotype had only moderate similarity to a range of Leptolyngbya species; and one phylotype was closely related to a number of Geitlerinema species. Generally the approaches yielded complementary information, however the results suggest that species designation based on morphological and ultrastructural criteria alone often fails to recognize their true phylogenetic position. Conversely some molecular techniques may fail to detect rare taxa suggesting that the widest possible suite of techniques be applied when conducting analyses of cyanobacterial diversity of natural populations. This is the first polyphasic evaluation of thermophilic cyanobacterial communities from the Australian continent.