Studies On The Allel Opathic Effects Of Macroalga Ulva Pertusa On Red Tide Microalgae And Isolation And Characterization Of Its Allelochemicals

In recent decades red tides (harmful microalgal blooms) occur frequently in both freshwater areas and marine coastal waters worldwide. This has caused disastrous damages to the fisheries and aquaculture industries, such as abalone breeding, prawn culture and caged fish culture. Because of the severe economic and public health problems caused by red tides, many studies have begun to concentrate on the control of undesired algal growth, and some promising methods have been developed including use of yellow loess, clay, copper sulfate and some biological agents, e.g. bacteria and viruses against red tides. Although these methods seem effective in some short-term experiments, they may have potentially dangerous environmental consequences.On the other hand, the appearance of excessive growth of green macroalgae in response to coastal eutrophication, (termed 'green tides' by marine ecologists) is an increasingly common phenomenon along shorelines worldwide. Blooms of green macroalgae have been reported to have a harmful impact on intertidal marine ecosystems in a variety of ways, including changes in ecosystem structure and reduction of biodiversity.The present studies deal with the allelopathic effects of the green macroalga Ulva pertusa on several red tide microalgae, and isolation and characterization of its allelochemicals. The three main objectives of these studies were to provide proof of the allelopathic effects of Ulva pertusa on these red tide microalgae, to gain an insight into the chemical interactions between macroalgae and microalgae in coastal areas, and to present evidence for the feasibility of using species of bloom-forming green macroalgae to control the proliferation of red tide microalgae.1. Comparative studies on the allelopathic effects of two different strains of Ulva pertusa on Heterosigma akashiwo andAlexandrium tamarensAllelopathic effects of fresh tissue and dry powder of a non-sexual and a sexual strain of the macroalga Ulva pertusa on the growth of the microalgae Heterosigma akashiwo and Alexandium tamarense were evaluated using long-term coexistence culture systems in which several concentrations of macroalga fresh tissue and dry powder were used. The effects of macroalga culture medium filtrate on the two HAB algae were also investigated. Moreover, isolation coculture systems were built to confirm the existence of allelochemicals and preclude the growth inhibition by direct contact. Short-term algicidal effect assays of macroalgae on H. akashiwo were carried out to measure the rate of algal cell lysis. The results of the coexistence assays showed that the growth of H. akashiwo and A. tamarense was strongly inhibited by fresh tissue and by dry powder of both strains of U. pertusa. The allelochemicals were lethal to H. akashiwo at relatively higher concentrations. The macroalga culture medium filtrate exhibited no apparent growth inhibitory effect on the two HAB algae under initial or semi-continuous filtrate addition, which suggested that continuous release of small quantities of rapidly degradable allelochemicals from the fresh tissue of both strains of U. pertusa was essential to effectively inhibit the growth of H. akashiwo and A. tamarense.2. Allelopathic growth inhibition of Prorocentrum mi cans (Dinophyta) by Ulva pertusa and Ulva linza (Chlorophyta) inlaboratory culturesAllelopathic effects of several concentrations of fresh tissue and dry powder of two bloom-forming green macroalgae Ulva pertusa and Ulva linza on the red tide microalga Prorocentrum micans were investigated in coexistence culture systems. Preliminary studies on the algicidal effects of one aqueous and four organic solventextracts from the macroalgae on the microalga were carried out to confirm the existence of allelochemicals in the tissue of these two macroalgae. The effects of filtrate of used macroalgal culture medium were tested on P. micans using a single initial or semi-continuous filtrate addition. Furthermore, the potential effects of the microalga on these two macroalgae were also evaluated. The results of the coexistence experiments demonstrated that the growth of P. micans was strongly inhibited by the addition of both the fresh tissue and the dry powder of both species of Ulva. Aqueous and methanol extracts of both macroalgae had strong growth inhibitory effects on P. micans, while the other three organic solvent extracts (acetone, ether and chloroform) had no apparent effect on its growth. This suggests that the allelochemicals from both macroalgae had relatively high polarities. The filtrate of used macroalgal culture medium exhibited no apparent growth inhibitory effect on the microalga under initial filtrate addition whereas the growth of P. micans was significantly inhibited under semi-continuous filtrate addition, which suggested that continuous release of small quantities of rapidly degradable allelochemicals from the fresh tissue of both macroalgae was essential to effectively inhibit the growth of/! micans. In contrast, the microalga had no apparent effect on the growth of either macroalgal species in coexistence experiments.3. Growth inhibition of three red tide microalgae by theextracts of Ulva pertusaGrowth inhibitory effects of different concentrations of distilled water extracts and 4 organic solvent (methanol, acetone, ether and chloroform) extracts of a nonsexual strain of Ulva pertusa on three typical red tide microalgae {Heterosigma akashiwo, Alexandrium tamarense and Prorocentrum micans) were investigated. Growth of the three microalgae was significantly inhibited by the distilled water extracts of U. pertusa at relatively higher concentrations while cells of the three microalgae did not die completely even at the highest concentration. Methanol extracts of U. pertusa showed the highest growth inhibitory activity on the three microalgae, and they killedall the cells of the three microalgae at relatively higher concentrations. The other 3 organic solvent extracts of U. pertusa had no apparent effect on the three microalgae, which suggested that the growth inhibitory substances from the extracts of U. pertusa had relatively high polarities. H. akashiwo was the most sensitive one while A. tamarense was the most tolerant one to the growth inhibitory substances from the extracts of U. pertusa.4. Isolation and characterization of a Ilelochemicals in thetissue of Ulva pertusa3.85 kg dry powder of Ulva pertusa was extracted with methanol, and 320 g extracts was obtained. The extracts were partitioned to petroleum ether phase, ethyl acetate phase, butanol phase and distilled water phase by liquid-liquid fractionation. Thereafter, the 4 phases were analyzed by HPLC, and petroleum ether phase and ethyl acetate phase were analyzed by GC/MS. Bioassays of the activity of every fraction were carried out on Heterosigma akashiwo. Petroleum ether phase and ethyl acetate phase had strong algicidal effect on the microalga. The results of GC/MS revealed that there were 16 fatty acids in the 2 phases, and most of them were unsaturated fatty acids. The results of bioassays demonstrated that 5 unsaturated fatty acids in the tissue of Ulva pertusa had strong algicidal effect on Heterosigma akashiwo, and the effective concentrations were below 5 mg I"1. The results of this study demonstrated that unsaturated fatty acids in the tissue of Ulva pertusa might be one of the components of the allelochemicals in the macroalga.5. Influence of non-nutrient environmental factors on the effectiveness of allelopathic effect of Ulva pertusa onHeterosigma akashiwo and their growthVarious effects of two-factor combinations of different levels of temperature (10, 15, 25 and 30°C), salinity (10, 20, 30 and 40 ppt), irradiance (20, 100, 200 and 400 umol m'2 s"1) and pH (5.5, 7, 8.5 and 10) on the effectiveness of allelopathic effect of Ulvapertusa on Heterosigma akashiwo and on their growth were investigated. Non-linear regression equations of the mean growth rates of Ulva pertusa and Heterosigma akashiwo, and of the microalgal growth inhibition rates by macroalga on two-factor combinations of the four environmental factors were obtained. Two-factor combinations of different levels of temperatures, salinity, irradiance and pH had significant (p<0.0l) influence on the mean growth rates of Ulva pertusa and Heterosigma akashiwo, and on the microalgal growth inhibition rates by macroalga. The optimal growth conditions for Ulva pertusa was: 20—25 °C, 30 ppt, 200—400 Hmol m"2 s"1 and pH=8.5 —10, while those for Heterosigma akashiwo was: 25°C, 30 ppt, 100 |amol m"2 s"1 and pH=8.5. The effectiveness of allelopathic effect of Ulva pertusa on Heterosigma akashiwo was increased by appropriate temperature (25 °C), low salinity (10 ppt), high irradiance (400 umol m'2 s"1) and high pH (10). Quadratic regression equations appropriately reflected the relationship between the three dependent variables above and the four environmental factors.6. Influence of different N, P nutrient levels on the effectiveness of allelopathic effect of Ulva pertusa onHeterosigma akashiwoAllelopathic effectiveness of Ulva pertusa of four different nutrient levels (N, P sufficient; N limitation; P limitation and N, P double limitation) on Heterosigma akashiwo of the same four different nutrient levels was investigated. Different N, P nutrient levels had significant influence on the effectiveness of allelopathic effect of Ulva pertusa on Heterosigma akashiwo. The allelopathic effectiveness was decreased when Ulva pertusa was under N, P double limitation. Cells of Heterosigma akashiwo under N limitation were more tolerant to the macroalga, while microalgal cells under N, P double limitation were more sensitive to the macroalga. Different allelopathic effectiveness of macroalgae on microalgae under different nutrient levels may be one of the mechanisms that caused different population dynamics of macroalgae and microalgae during the change of N, P nutrient in coastal areas.