Culture solutions of the submerged freshwater macrophytes Hydrilla verticillata and Vallisneria spiralis, separately incubated at 10 g of fresh weight (FW) per liter for three days, were extracted by solid phase extraction (SPE) followed by liquid liquid extraction (LLE), and the acquired ethyl acetate fractions were analyzed by gas chromatograph-mass spectrometry (GC-MS) to identify potential allelochemicals exuded from these plant species. Freeze-dried plant tissues were separately methanol-extracted with a similar LLE-GC-MS procedure to compare allelochemical production and exudation. Four phenolic compounds were identified for both species: vanillic acid (VA), protocatechuic acid (PA), ferulic acid (FA), and caffeic acid (CA). H.verticillata produced 179 times the amount of phenolic compounds of V. spiralis. The phenolic contents in the culture solution were lower than 10% of those in the plant tissues for both submerged species in six of the eight cases. When evaluating the joint effects of the compounds using the Toxicity Index (TI) model, it was observed that the four phenolic compounds exerted additive and synergistic inhibition effects on the growth of Microcystis aeruginosa depending on the mixing ratios. These results indicate that H. verticillata and V. spiralis could release some phenolic allelochemicals to inhibit the growth of M. aeruginosa, and the joint action of multiple allelochemicals may be an important allelopathic pattern of submerged macrophytes to inhibit the growth of noxious cyanobacteria in natural aquatic ecosystems.
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