Allelopathic Effects Of Two Common Diatoms And The Preliminary Research Of Chaetoceros Curvisetus Allelochemicals

Phytoplankton allelopathy in the marine environment has been paid more and more attention with the frequent occurrence of red tides, and numerous researches have proved allelopathy is essential to the formation and maintenance of blooms. Allelopathic substance is the media by which phytoplankton constrains or promotes the coexisting species, which is significant in the research of allelopathy. However, apart from the paucity of allelopathy knowledge among phytoplankton in marine ecosystems, the property and structure about most allelochemical remain unknown. Moreover, the investigations about the marine phytoplankton allelopathic compounds have almost been concerned with chrysophyta and dinoflagellate, and few investigations of allelopathic compounds are about diatom. Skeletonema costatum and Chaetoceros curvisetus are common red tide species in coastal area of China, and they are used as target species and donor species during our investigation. A series of algal cultures and compound extractions are conducted to research the allelopathic effect between the two diatoms and the properties and structure of C. curvisetus allelochemical.First, the means of cell-free filtrate culture were used to explore the allelopathic effect between the two diatoms. The results showed that cell-free filtrated culture of C. curvisetus in the later exponential growth phase inhibited the growth of S. costatum significantly in enriched nutrients conditions, and the cell size of S. costatum was affected as well. On the contrary, there was no obvious influence on the relative growth rate and the cell size of C. curvisetus by enriched S. costatum cell-free filtrates in the end of exponential growth phase, which indicated that S. costatum filtrate showed no allelopathic effects on C. curvisetus. The results proved the the presence of the allelopathy between marine diatoms.Secondly, on the basis of the fact that Chaetoceros curvisetus showed allelopathic effect on Skeletonema costatum, a series of experiments, including algal cultures and compounds extraction, were carried out to research the properties, influence factors, and degradation characteristics of C. curvisetus allelochemical. The results indicated the filtrates of C. curvisetus in exponential phase showed stronger inhibitory effect than that in declining phase filtrates. C. curvisetus allelochemical remained stable when the temperature was lower than 50 ℃. The allelopathic substance degraded attributed to light and bacteria and bacteria contributed more. The ethyl acetate extraction of C. curvisetus filtrates showed significant allelopathic effect and the wavelength of characteristic absorption for the growth inhibitor was 255-260nm. Moreover, a mathematical model was established to simulate the relationship between the average inhibition rate of S. costatum and the concentration of C. curvisetus allelochemical in this research, and the theoretical model agreed well with the experimental results. Consequently, the allelochemical of C. curvisetus was supported to be benzene derivatives whose decomposition temperature was more than 50 ℃. The study provided experimental basis for extracting allelochemical in the further study.Thirdly, the bioactive compounds of C. curvisetus were extracted from dried algae cell, on the premise that the allelochemical was stable, to explore the effect of phenols, terpenes, polysaccharides and alkaloids crude extracts on the growth of S. costatum. The results indicated that the phenols, terpenes, alkaloids extract inhibited the growth of S. costatum. The polysaccharide extracts had no effect on the growth of S. costatum. According to the comparison of S. costatum growth curves and the change of cell morphology in the crude extract and cell-free filtrate, the results suggest that the active substance of C. curvisetus working on S. costatum might be alkaloids. This experiment examined the possible type of C. curvisetus allelochemical which showed the depressing effect to S. costatum.Fourthly, the experiments to explore the extract conditions and the examine of biological effects were conducted to separate and enrich the C. curvisetus allelochemical in the cell-free filtrate, and the structure of substance were identified. The results showed that a small-volume culture and easy counting methods can be applied to explore the allelopathic effect of separation component. The separation results of Si-SPE columns on the C. curvisetus filtrate were favorable, and ethyl acetate was used as the eluent. Sephadex-25 columns worked well on the separation of the ethyl acetate component of C. curvisetus filtrate, and the Mili-Q water was used as eluent. And then the F-Ⅱ component separated by Sephadex-25 columns was injected into C18 column, and the component with a retention time of 10-14min was selected to identify the structure. The molecular weight of C. curvisetus allelochemical might be 314, and the allelochemical might be 2-((2-cyanophenyl) amino)-2-oxoethyl,3-cyclohexyl propanoate.In summary, this study investigated the phenomenon and property of allelopathy between C. curvisetus and S. costatum systematically and a set of complete method to extract, separate and enrich the allelochemical from the filtrate was established. The effective allelopathic substances were selected and identified as well. Since the chemical composition of the filtrate is complex and the concentration of allelochemical is at a low level, the purification of allelochemical is always interfered by other substances. Further research should be carried out to enrich and identify the definite structure of the allelochemial in a large-volume filtrate in the future. The research is significant in evaluating the effect of allelopathy on the competition of marine phytoplankton species and on the formation and development of blooms, which can provide reference for the bioremediation of the red tides.