Young-Tae Park

Development of a SYTO9 Based Real-Time PCR Probe for Detection and Quantification of Toxic Dinoflagellate Karlodinium Veneficum (Dinophyceae) in Environmental Samples

T.-G. Park, Y.-T. Park and Y. Lee. 2009. Development of a SYTO9 based real-time PCR probe for detection and quantification of toxic dinoflagellate Karlodinium veneficum (Dinophyceae) in environmental samples. Phycologia 48: 32–43. DOI: 10.2216/08-52.1. Conventional methods to identify the fish-killing dinoflagellate Karlodinium veneficum rely on rDNA sequence and scanning electron microscopy analyses that are not suitable for processing a large number of samples. To overcome this difficulty, SYTO9 and TaqMan format real-time PCR probes were developed for the internal transcribed spacer rDNA region for rapid detection and quantification of K. veneficum in the marine environment. Assay specificity, sensitivity, and accuracy were confirmed by testing against related organisms, sequencing PCR amplicons from field samples, and comparing real-time PCR results with microscopic counts. The SYTO9-based assay produced highly reproducible DNA melting curves over a broad range of starting DNA template. When the SYTO9- and TaqMan-based assays were compared in the quantitative measurements, they showed comparable results and reasonable reproducibility. The K. veneficum–specific assays were used in conjunction with Pseudopfiesteria shumwayae (Dinophyceae)–, Pfiesteria piscicida (Dinophyceae)–, and Cryptoperidiniopsis brodyi (Dinophyceae)–specific real-time PCR probes to investigate temporal changes in abundances of these four species in Chinhae Bay, South Korea. The field survey revealed that K. veneficum was dominant (maximum 970 cells ml−1) among those species; whereas, Pfiesteria species rarely occurred. These findings suggest that the SYTO9- and TaqMan-based assays are specific and sensitive for detecting and quantifying K. veneficum in the environment. The wide distribution of K. veneficum also indicates potential fish kills by this organism in Chinhae Bay.

Detection of Fish Killing Dinoflagellates Cochlodinium polykrikoides and Karlodinium veneficum (Dinophyceae) in the East China Sea by Real-time PCR

The rDNAs of figh-killing dinoflagellates Cochlodinium polykrikoides and Karlodinium veneficum were detected from the East China Sea by species-specific real-time PCR probes. Sequence analysesusing the partial ITS sequences from the real-time PCR products showed identical sequences with C. Polykrikoides and K. veneficum, respectively and low expectation values (E-value) of less than 1e-5 suggesting the presence of these organisms in the East Ching Sea shelf water that flows into the Tsushima Strait and the Yellow Sea.

Effects of Yellow Clay on the Production of Volatile Fatty Acids during the Anaerobic Decomposition of the Red Tide Dinoflagellate Cochlodinium polykrikoides in Marine Sediments

The formation of volatile fatty acids(VFAs) and changes in pH, oxidation and reduction potential(Eh) and acid volatile sulfide(AVS) with the addition of yellow clay were investigated using microcosm systems to examine the effects of yellow clay dispersion on the anaerobic decomposition of Cochlodinium polykrikoides in marine sediments. The acetate concentration reached a maximum by day 4 and was 1.2-1.8 fold less in the sample treated with yellow clay compared to the untreated sample (224-270 vs. 333 uM). The formate concentration reached a maximum by day 1 and was 1.3-2.8 fold less in the sample treated with yellow clay compared to the untreated sample (202-439 vs. 563 uM). The propionate concentration reached a maximum by day 2 and was 1.5-1.8 fold less in the sample treated with yellow clay compared to the untreated sample (32.6 vs. 57.2 uM). After the amounts of acetate, formate and propionate peaked the levels dropped dramatically due to the utilization by sulfate reducing bacteria. The Eh of the samples treated with yellow clay was similar to the untreated sample on day 0 but was higher in the sample treated with yellow clay(140-206 mV) from days 4 to 17. AVS started to form on day 3 and this was sustained until day 6, and 1.2-2.2 fold less was produced in the sample treated with yellow clay compared to the untreated sample (40.2-69.3 vs. 83.2-93.8 mg/L). Accordingly, during the anaerobic decomposition of C. polykrikoides in marine sediments, yellow clay dispersal seems to suppress the reduction state of Eh and the formation of volatile fatty acids(acetate, formate and propionate) used as an energy source by sulfate reducing bacteria, indicating that this process controls the production of hydrogen sulfide that negatively affects marine organisms and the marine sediment environment.