Allelopathic Effects And Mechanism Of Fructus Ligustri Lucidi Extract On Microcystis Aeruginosa

With the eutrophication of fresh water worldwide increased,heavy cyanobacterial blooms break out frequently;Microcystis Aeruginosa bloom is the most common one.To find safe and reliable,cheap and efficient bloom control method has been a hot topic in the field of ecological protection.The allelopathy of terrestrial plants,with the wide sources and large biomass of raw material,high efficiency,easy controlling and low toxicity,become a potential new algal inhibiting method.This research took the dominant species-M.Aeruginosa as the object,systematically studied allelopathic effects of Fructus ligustri lucidi extract(FLL)on M.Aeruginosa and its mechanism.Firstly,we determined the algal inhibition effect of FLL by a series of tests,and established the allelopathic inhibitory model.Then,the allelochemical was screened out and its structure was identified by separation and activity tests.Meanwhile,we investigated the inhibition mechanism from the cell physiology and morphology perspective.The main theses of this research were as follows:1.The inhibition effect of FLL at five dosages(0.25,0.5,1.0,2.0,4.0g·L-1)on M.Aeruginosa was investigated.The assays found:(1)The cell density,Chl-a fluorescence and metabolic activity of cells declined gradually in short time in all treated groups.During 25-d incubation time,the cells could not be detected in the treatment groups with 2.0 g·L-1 and 4.0 g-L-1 dosages.(2)The propidium iodide(PI)-staining and transmission electron microscopy(TEM)assays found that the cell membranes were broken after 1-d incubation,and were proportional to time and dosage.(3)FLL at 0.25 g·L-1 was more like algistatic while FLL at 0.5 g·L-1 or above had algicidal property.The results suggested that,FLL could damage the cell membrane,hamper the chlorophyll synthesis and division growth,inhibit the activity of esterase in M.Aeruginosa cells,that might be the main reason why the cell growth was inhibited.2.The allelopathic effect and nutrient effect of FLL was investigated,and allelopathic inhibition model was established.The anti-algae experiment was conducted by adding FLL with different pretreatment methods and gradient concentrations.Orthogonal experimental results showed that the pretreatment methods of FLL had great influence on algal inhibiting effect,The pretreatment method leading to the best anti-algae effect(EC50=0.344)was:anaerobic fermentation in the light after sterilization.Gradient tests showed that FLL from different pretreatment methods had different nutrient effect on algae.The nutrient effect of FLL which was at low concentration in sterilization group was weak,while the effect of FLL which was at high concentration in no sterilization group was stronger.A toxicological database was established with eight different groups of leaching,which were treated with different concentrations in six different groups,and went on non-linear fitting with the combination of An-Johnson-Lovett hormesis model and Logistic growth model,and the model could fit the data in general.3.The initial separation of allelopathic substance in FLL was conducted by the combination of extraction separation and active track.PEth and CHCl3 fractions could stimulate cell growth instead inhibiting it;the EtOAC fraction had weak influence on inhibition;after 15-d incubation,the inhibition rate of BuOH and water fractions were 97.14%and 100%,respectively.The removal efficiency of Chl-a,PC,APC and PE in BuOH and water groups was over 80%,the Ca2+Mg2+-ATPase activity decreased,POD and CAT activity showed obvious increase,SOD activity raised at first,then decreased.The enzyme activity could not be detected after 10-d incubation in water group.The results illustrated that BuOH and water groups could inhibit the synthesis of photosynthetic pigments,lead calcium loss of the mitochondria and lipid peroxidation,which could cause cell membrane damage.PI-staining test showed that BuOH and water fractions could break the cell membrane,their extracts could be algicidal agent.4.The main allelochemicals were isolated step by step from FLL,using multi chemical analysis methods of plant natural products chemistry.The results showed that the inhibition rate of No.7-1 subdivision fraction from water fraction was nearly 100%,the structures was identified to be C6H14O6,namely D-mannitol.D-mannitol is a kind of natural sugar alcohols,as a high osmotic dehydrant in medicine;the high osmotic dehydration effect of D-mannitol on M.Aeruginosa might be the key factor in algal inhibition.The inhibition rate of No.4 coarse fraction from BuOH fraction was 100%,but the inhibition rates of its subdivision fractions were not high,which meant the result was from the synergistic reaction among many components,the mechanism has to be studied further.