| Severe outbreaks of harmful algal blooms (hereafter, HABs) cause serious problems with regard to effective utilization of water resources such as fisheries and water-supply reservoirs, moreover, about 2000 cases of human poisoning resulting from algal toxin are reported every year. Because of the severe economic and public health problems caused by harmful microalgae, many studies about the blooms of harmful microalgae have been conducted. Some promising methods have been developed including the use of yellow losses to sediment red tide organisms, chemical agents such as copper sulfate, hydrogen peroxide, and some biological controls in the form of viruses or bacteria. Although these methods seem effective in some short-term experiments, they may have potentially dangerous environmental consequences; therefore there are very limited investigations of a direct and specific control of marine harmful algal blooms with few environmental side-effects.To look for HABs control agents that are efficient and benign to the environment, many scientists show a growing interest for growth inhibition of HAB species by allelopathic substances released by other aquatic organisms. Macroalgae and microalgae were known to have an antagonistic relationship in both natural and experimental aquatic ecosystems.The present studies deal with the allelopathic effects of three macroalgae Ulva linza (Chlorophyta), Grateloupia turuturu (Rhodophyta) and Sargassum thunbergii (Phaeophyta) on the red tide microalga Prorocentrum donghaiense, and isolation and characterization of these allelochemicals. The three main objectives of these studies were to provide proof of the allelopathic effects of three macroalgae on P. donghaiense, to gain an insight into the chemical interactions between macroalgae and microalgae in coastal areas, and to present evidence for the feasibility of using many macroalgae to control the growth of red tide microalgae. Results show that:1. Comparative studies on the allelopathic effects of three macroalgae on P. donghaienseWe conducted laboratory experiments under stable environmental conditions, precluding the nutrient, pH, light conditions and effects of bacteria in order to investigate the allelopathic effects of fresh tissue, dry powder and aqueous extracts of three macroalga U. linza, G. turuturu and S. thunbergii on the growth of microalga P. donghaiense were evaluated using coexistence culture systems in which concentrations of three macroalga were varied. The effects of macroalga culture medium filtrate on P. donghaiense were also investigated. The results coexistence assay showed that the growth of P. donghaiense was strongly inhibited by using fresh tissues and dry powder; this indicated that there are the allelopathic effects of three macroalgae on P. donghaiense. Aqueous extracts of various concentrations of three macroalga can inhibit the growth of P. donghaiense and indicated that there are allelochemicals in three macroalgae; The effects of the macroalga culture medium filtrate exhibited apparent inhibitory effect both under initial filtrate addition and semi-continuous addition, which suggested that release of allelochemicals from the fresh tissue of macroalga were essential to effectively inhibit the growth of P. donghaiense. In our study, we also concluded that the allelopathic effects of macroalgae on P. donghaiense show universality and species-specific.2. Growth inhibition of P. donghaiense by the extracts of U. linza, G. turuturu and S. thunbergiiGrowth inhibitory effects of different concentrations of distilled water extracts and 4 organic solvent (methanol, acetone, ether and chloroform) extracts of U. linza, G. turuturu and S. thunbergii on the microalgae P. donghaiense were investigated. Methanol extracts of the highest growth inhibitory activity on two microalgae, and they killed all the cells of P. donghaiense at relatively higher concentrations. Growth of P. donghaiense was significantly inhibited by the distilled water extracts of three macroalgae at relatively higher concentrations while cells of P. donghaiense did not die completely even at the highest concentration. The other 3 organic solvent extracts of three macroagae had no apparent effect on P. donghaiense, which suggested that the growth inhibitory substances from the extracts of three macroalgae had relatively high polarities. The methanol extracts of U. linza, G. turuturu were partitioned to petroleum ether phase, ethyl acetate phase, butanol phase and distilled phase by liquid-liquid fractionation. The bioassays of the activity of every fraction were carried out on P. donghaiense, indicated that petroleum ether phase and ethyl acetate phase had strong algicidal effect on the microalgae, fatty acids are probably main allelochemicals in the tissue of macroalgae.3. Influence of temperature, salinity, irradiance and pH on the effectiveness of allelopathic effects of U. linza, G. turuturu on P. donghaienseTwo-factor combinations of different levels of temperature (10, 15, 25 and 30), salinity (10, 20, 30 and 40), irradiance (20,100,200 and 400μmol m-2s-1) and pH (5.5, 7, 8.5 and 10) on the effectiveness of allelopathic effect of U. linza, G. turuturu on P. donghaiense were investigated. Two-factor combinations of different levels of temperature, salinity, irradiance and pH had significant (p<0.01) influence on P. donghaiense growth inhibition rates by U. linza, G. turuturu. The effectiveness of allelopathic effects of U. linza, G. turuturu on P. donhaiense was increased by low temperature (15°C), low salinity (20), high irradiance (400μmol m-2s-1) and high pH (10).
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