Myriophyllum spicatum-released allelopathic polyphenols inhibiting growth of blue-green algae Microcystis aeruginosa
Introduction
With regard to the antagonistic relationship occurring between algae and macrophytes in natural and experimental aquatic ecosystems (Hasler and Jones., 1949), the competition for available nutrients and light is generally known to inhibit algal growth. Hogetsu et al. (1960) proposed another mechanism, in which macrophytes release allelochemicals that inhibit algal growth. Such a growth inhibition mechanism strongly suggests that macrophytes could be used to control undesirable algae. We recently investigated the allelopathic effects produced by nine species of macrophytes (Nakai et al., 1999) and found that Myriophyllum spicatum produced the most inhibitory effects on two species of problem-causing blue-green algae (Microcystis aeruginosa and Phormidium tenue). Moreover, the inhibitory effects of M. spicatum were demonstrated to be due to its release of allelochemicals, thereby confirming the feasibility of using macrophytes as a control tool for algal growth.
Planas et al. (1981) found that an extract of M. spicatum—which included 12 kinds of phenols and polyphenols, e.g., gallic acid and ellagic acid—could inhibit algal growth. They did not, however, investigate the inhibitory effects produced by each individual compound. Regarding other species of the genus Myriophyllum, Saito et al. (1989) showed that the growth of blue-green algae Anabaena flos-aquae and M. aeruginosa could be inhibited by the hydrolyzable tannins eugeniin and 1-desgalloyl eugeniin extracted from M. brasiliense and as well by gallic and ellagic acids, which are components of these hydrolyzable tannins. Later, Aliotta et al. (1992) extracted three polyphenols from M. verticillatum and confirmed the associated inhibitory effects on algal growth.
Now, in view of the fact that phenols, and especially polyphenols, have high water solubility due to their hydroxyl groups, and that M. spicatum contains phenols and polyphenols (Planas et al., 1981), it is reasonable to surmise that M. spicatum releases phenols and/or polyphenols which cause the resultant growth inhibition of algae. In fact, Gross et al. (1996) showed that M. spicatum released tellimagrandin II and ellagic acid, and that each compound produced an inhibitory effect. Questions remain, though, because the amounts released by M. spicatum have not been evaluated, and some phenolic compounds released by it have not been identified.
Although the feasibility of controlling algal growth by the addition of allelopathic macrophyte M. spicatum and/or its allelochemicals has been demonstrated, further research must be carried out to establish this as a safe, effective method for aquatic ecosystem management. Essential tasks are (1) to reveal the allelopathic compounds released from M. spicatum, (2) to determine a quantitative relationship that clarifies how the inhibition of algal growth is affected by the concentration of each allelochemical, and (3) to study collective activity of the allelochemicals on algal growth inhibition. Toward this end, here we identify the allelochemicals released by M. spicatum and report on a quantitative investigation of the growth inhibitory effects of the identified allelochemicals. In addition, collective activity of the identified allelochemicals and their inhibitory effects were investigated to determine whether or not the identified allelochemicals demonstrate synergistic growth inhibition of algae.
Section snippets
Algae and M. spicatum
As one of the most undesirable blue-green algae in Japan, Microcystis aeruginosa (NIES-87) obtained from the microbial collection of the National Institute for Environmental Studies (NIES), Japan was used for algal assays. M. spicatum was collected from the Asakawa River, Tokyo, Japan and then cultivated in a 20-fold dilution Gorham’s medium (Zehnder and Gorham, 1960) using a light intensity of 3000 lux at 25°C for 3 days.
Algal assays were used to: (1) accomplish assay-directed fractionation of
Identification of allelochemicals
Figure 2(a) shows the resultant HPLC chromatogram of the UWF. Comparing retention time of the occurred peaks with that of authentic standards, peak 1 at 11.89 min, 2 at 16.44 min, 3 at 48.25 min, 4 at 87.10 min are considered to indicate polyphenols, i.e., pyrogallic acid (PA), gallic acid (GA), (+)-catechin (CATECH), and ellagic acid (EA) whose structures are shown in Fig. 3, however many unknown peaks are presented. Tellimagrandin II (Gross et al., 1996) is thought to elute between GA (peak
Conclusions
Using HPLC and APCI-MS analysis, we found that (1) macrophyte M. spicatum releases four polyphenols, i.e., PA, GA, CATECH and EA, each of which inhibits the growth of blue-green algae M. aeruginosa, and (2) the autoxidized products of these polyphenols also produce growth inhibition effects. A quantitative investigation of their respective inhibitory effects on the maximum growth of M. aeruginosa showed that PA and GA produce strong inhibitory effects compared to those of CATECH and EA which
Acknowledgements
We are grateful to the National Institute for Environmental Studies, Japan, for supplying M. aeruginosa.
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