Abstract
The volatile metabolite, 2-Methylisoborneol (2-MIB) produced by cyanobacterial species, causes odor and taste problems in freshwater systems. However, simple identification of cyanobacteria that produce such off-flavors may be insufficient to establish the causal agent of off-flavor-related problems as the production-related genes are often strain-specific. Here, we designed a set of primers for detecting and quantifying 2-MIB-synthesizing cyanobacteria based on mibC gene sequences (encoding 2-MIB synthesis-catalyzing monoterpene cyclase) from various Oscillatoriales and Synechococcales cyanobacterial strains deposited in GenBank. Cyanobacterial cells and environmental DNA and RNA were collected from both the water column and sediment of a eutrophic stream (the Gong-ji Stream, Chuncheon, South Korea), which has a high 2-MIB concentration. Primer sets mibC196 and mibC300 showed universality to mibC in the Synechococcales and Oscillatoriales strains; the mibC132 primer showed high specificity for Pseudanabaena and Planktothricoides mibC. Our mibC primers showed excellent amplification efficiency (100–102%) and high correlation among related variables (2-MIB concentration with water RNA r = 689, p < 0.01; sediment DNA r = 0.794, p < 0.01; and water DNA r = 0.644, p < 0.05; cyanobacteria cell density with water RNA and DNA r = 0.995, p < 0.01). These primers offer an efficient tool for identifying cyanobacterial strains possessing mibC genes (and thus 2-MIB-producing potential) and for evaluating mibC gene expression as an early warning of massive cyanobacterial occurrence.
Highlights
Occurring 2-methylisoborneol (2-MIB) and geosmin produced by cyanobacteria and actinomyces are regarded as a worldwide problem in freshwater systems because of their muddy or earthy odorous properties [1,2]
To detect the 2-MIB-synthesizing gene mibC in cyanobacteria, eight sets of primers were constructed based on the sequence alignment of mibC genes from diverse cyanobacteria (Table 1)
Other primersdesigned designedin inthis thisstudy study. These unexpected non-specific amplicons mibC719) amplified non-specific amplicons during polymerase chain reaction (PCR). These unexpected non-specific amplicons could have been caused by non-specific binding [37], which could lead to overestimation of the 2could have been caused by non-specific binding [37], which could lead to overestimation of the 2-MIB
Summary
Occurring 2-methylisoborneol (2-MIB) and geosmin produced by cyanobacteria and actinomyces are regarded as a worldwide problem in freshwater systems because of their muddy or earthy odorous properties [1,2]. Cyanobacteria are recognized as the major producer of the unpleasant odorous 2-MIB; the odor is more severe during cyanobacterial blooms. Prior studies indicated that blooms of cyanobacteria coincided with the occurrence of odor substances in various freshwater systems [5,6], including Huajiang Lake in Mongol, where the maximum concentration of 2-MIB reached 102 ng/L during cyanobacterial blooms [7]. The major cyanobacterial genus in the North Han River in Korea has shifted from Anabaena to Pseudanabaena since 2013, accompanied by an increase in the occurrence and. Public Health 2020, 17, 1933; doi:10.3390/ijerph17061933 www.mdpi.com/journal/ijerph
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