Allelopathic Effect Of Enteromorpha Linza On Two Species Of Marine Microalgae And The Allelochemicals' Isolation And Characterization

Allelopathy is a widespread natural phenomenon in aquatic ecosystem, including the growth promotion and inhibition among various organisms. Allelopathy is one of the hot topics mainly because allelopathy between macroalgae and red tide microalgae is a potentially biological method on bloom controlling. On the other hand, some green-tide forming species along with the "green tide" caused by green algae (Ulva and Enteromorpha) have proved to affect the co-cultured microalgae, and the studies on their allelopathic effects can provide basis for effectively using these macroalgae and exploring the possible generation mechanism. Therefore, the research on the allelopathy between causative species of green tide and microalgae around them is of great practical significance. This present study dealed with the allelopathic effects of macroalgae(Enteromorpha linza) on two species of marine microalgae Prorocentrum minimum and Platymonas helgolandica under controlled laboratory conditions, and then isolated and characterized the allelochemicals that had inhibitory effects on microalgae, gaining an insight into the chemicals interactions between E. linza and two microalgae.Results showed that:1. The allelopathic effects of E. linza on P. minimum and P. helgolandica.The allelopathic experiments were conducted under controlled laboratory conditions to preclude the variation of the nutrient, pH, light conditions and environmental bacteria. The target marine microalgae were cultured with fresh tissue, dry powder, aqueous extracts, and culture medium filtrate of E. linza to set up microalga-macroalga co-culture. Results showed that:(1) The fresh tissue, dry powder of E. linza had significantly inhibitory effects on the growth of the co-cultured microalgae, and the inhibition raised along with the increase of the macroalgal concentrations (P<0.05). Lethal effect was even observed on P. minimum when the concentrations of E. linza were set at 5 and 10 gFW/L. However, the inhibitory effects declined gradually in the dry powder group with culturing time increasing. P. minimum and P. helgolandica's values of EC50 treated by fresh tissue were 1.41 gFW/L and 2.50gFW/L, respectively; P. minimum and P. helgolandica's values of EC50 treated by dry powder were 1.41 gFW/L and 2.50 gFW/L, respectively. From the values of EC50, the allelopathic effects of fresh tissue are stronger than dry powder, and compared to P. helgolandica, P. minimum is more sentitive to E. linza. (2) All the concentrations of aqueous extracts of E. linza had inhibitory effects on the growth of the two microalgae during the first 96 h (P<0.05), and little change was observed thereafter till the end of the experiment in most of treatments as compared to the control, inferring that the allelochemicals exist in the E. linza's tissue. (3) The culture medium filtrate of E. linza showed inhibition on the growth of P. minimum (P<0.05) while had little effect on P. helgolandica (P>0.05). It seemed that E. linza liberated allelochemical(s) into the environment to conduct the allelopathic effects.2 Inhibitory effects of the E. linza extracts on growth of P. minimum and P. helgolandicaThe methanol extracts of E. linza were partitioned to petroleum ether phase, ethyl acetate phase, butanol phase and distilled phase (the polarity followed by high to low) by liquid-liquid fractionation. The bioassays of the activity of every fraction were carried out on two microalgae, compared to the control groups which merely treated by the solvent, indicating that petroleum ether phase had strong allelopathic effect on the two microalgae (P<0.05), and the allelopathic effect ethyl acetate phase was weaker. The others almost had no allelopathic effects (P>0.05).3 Isolation and identification of allelochemicals in fresh tissue of E. linzaThe petroleum ether phase and ethyl acetate phase of the methanol extracts of E. linza were analyzed by Gas Chromatography/Mass Spectrometry (GC/MS). The results of GC/MS revealed that there were 17 fatty acids. The contents of unsaturated fatty acids of petroleum ether phase and ethyl acetate phase were 51.11% and 29.87%, and the contents of saturated fatty acids were 48.89% and 70.13%. The three most abundant fatty acids were 16 carbonate,9E-octadecenoic acid and 9Z,12Z, 15Z-octadecadienoic acid. According to these results, fatty acids may be the major component of allelochemicals in E. linza.