Aphanizomenon spp. have formed harmful cyanobacterial blooms in the Nakdong River during spring, autumn, and now in winter, and the expansion of blooming period and area, associated with the global warming is predicted. The genus Aphanizomenon has been described to produce harmful secondary metabolites such as off-flavors and cyanotoxins. Therefore, the production of harmful secondary metabolites from the Aphanizomenon blooms in the Nakdong River needs to be monitored to minimize the risk to both water quality and public health. Here, we sampled the cyanobacterial blooms in the Nakdong River and isolated ten Aphanizomenon strains, morphologically classified as Aphanizomenon flos-aquae Ralfs ex Bornet et Flahault 1888. Phylogenetic analysis using 16S rRNA and internal transcribed spacer (ITS) region nucleotide sequences confirmed this classification. We further verified the harmful secondary metabolites-producing potential of A. flos-aquae isolates and water samples containing cyanobacterial blooms using PCR with specific primer sets for genes involved in biosynthesis of off-flavor metabolites (geosmin) and toxins (microcystins, saxitoxins and cylindrospermopsins). It was confirmed that these metabolite biosynthesis genes were not identified in all isolates and water samples containing only Aphanizomenon spp. Thus, it is likely that there is a low potential for the production of off-flavor metabolites and cyanotoxins in Aphanizomenon blooms in the Nakdong River.

The geographical range of invasive cyanobacteria with high toxigenic potential is widening because of eutrophication and global warming, thus, monitoring their appearance is necessary for safe water quality control. Most invasive cyanobacteria are nostocalean species, and their accurate identification by classical morphological methods may be problematic. In this study, we developed polymerase chain reaction (PCR) primers to selectively identify five invasive cyanobacterial genera, namely, Chrysosporum, Cuspidothrix, Cylindrospermopsis, Raphidiopsis, and Sphaerospermopsis, using genetic markers such as rbcLX, rpoB, rpoC1, and cpcBA, and determined the amplification conditions for each pair of primers. The primer performances were verified on single or mixed nostocalean cyanobacterial isolates. The five primers allowed selective identification of all the target genera. In field samples collected during summer, when cyanobacteria flourished in the Nakdong River, the respective PCR product was observed in all samples where the target genus was detected by microscopic analysis. Besides, weak bands corresponding to Sphaerospermopsis and Raphidiopsis were observed in some samples in which these genera were not detected by microscopy, suggesting that the cell densities were below the detection limit of the microscopic method used. Thus, the genus-specific primers developed in this study enable molecular monitoring to supplement the current microscopy-based monitoring.

Invasive nostocalean cyanobacteria (INC) were first reported in tropical regions and are now globally spreading rapidly due to climate change, appearing in temperate regions. INC require continuous monitoring for water resource management because of their high toxin production potential. However, it is difficult to analyze INC under a microscope because of their morphological similarity to nostocalean cyanobacteria such as the genus Aphanizomenon. This study calculates the gene copy number per cell for each target gene through quantitative gene analysis on the basis of genus-specific primers of genera Cylindrospermopsis, Sphaerospermopsis, and Cuspidothrix, and the toxin primers of anatoxin-a, saxitoxin, and cylindrospermopsin. In addition, quantitative gene analysis was performed at eight sites in the Nakdong River to assess the appearance of INC and their toxin production potential. Genera Cylindrospermopsis and Sphaerospermopsis did not exceed 100 cells mL⁻¹ at the maximum, with a low likelihood of related toxin occurrence. The genus Cuspidothrix showed the highest cell density (1759 cells mL⁻¹) among the INC. Nakdong River has potential for the occurrence of anatoxin-a through biosynthesis by genus Cuspidothrix because the appearance of this genus coincided with that of the anatoxin-a synthesis gene (anaF) and the detection of the toxin by ELISA.

The proliferation of harmful cyanobacteria, particularly Microcystis, poses significant risks to drinking and recreational water resources, especially under the influence of climate change. Conventional monitoring methods based on microscopy for harmful cyanobacteria management systems are limited in detecting toxigenic genotypes, hindering accurate risk assessment. In this study, we developed a digital droplet PCR (ddPCR)-based method for the simultaneous quantification of total and toxigenic Microcystis in freshwater environments. We targeted the secA gene, specific to the Microcystis genus, and the mcyA gene, associated with microcystin biosynthesis. Custom-designed primers and probes showed high specificity and sensitivity, enabling accurate detection without cross-reactivity. The multiplex ddPCR assay allowed for concurrent quantification of both targets in a single reaction, reducing the analysis time and cost. Application to field samples demonstrated good agreement with microscopic counts and revealed seasonal shifts in toxigenic genotype abundance. Notably, ddPCR detected Microcystis at very low densities—down to 7 cells/mL in the mixed cyanobacterial communities of field samples—even when microscopy failed, highlighting its utility for early bloom detection. This approach provides a reliable and efficient tool for monitoring Microcystis dynamics and assessing toxin production potential, offering significant advantages for the early warning and proactive management of harmful cyanobacterial blooms.