Inhibitory Effect Of Formula Algal Suppressor Preparations From Common Terrestrial Plants On Prorocentrum Donghaiense

Since the 21st century,with the increasing harmful algal blooms（HABs）to Marine ecological environment,the prevention and control of HABs has gradually received extensive attention.Among them,Prorocentrum donghaiense is the main cause of HABs in our country.Its outbreak seriously endangers the aquaculture industry and offshore ecological environment,so an effective control method is urgently needed.At present,many control methods of HABs have different defects,but the allelopathy of plants to control HABs has the advantages of low cost,high efficiency,no secondary pollution and little ecological impact,so it is expected to be a promising control method of HABs.In order to find an effective means to control HABs,Cinnamomum camphora and Ginkgo biloba leaves were used to explore the inhibitory characteristics of P.donghaiense.Based on the literature,the main allelopathic components of the extracts from C.camphora and G.biloba leaves were deduced and tested respectively.Then,a compound with high content and strong algal inhibition effect was screened from C.camphora leaf extract,and the compound was combined with G.biloba leaf extract to form a formula algal suppressor.Then explore the inhibitory mechanism of the formula algal suppressor on P.donghaiense,and detect the ecological safety of the formula.The main conclusions of this study are as follows:（1）The effects of sea water extracts of C.camphora and G.biloba on the inhibition rate and motility of P.donghaiense were studied.The results showed that the inhibitory effect of C.camphora extract was slow,and there was no significant difference between the experimental group and the control group within 48 h of treatment（P>0.05）.After72 h of treatment,the experimental group with the concentration higher than 1.0 g/L had a significant inhibitory effect on the growth of P.donghaiense（P<0.05）.High concentration of C.camphora extract（≥3.0 g/L）was able to rupture a large number of P.donghaiense cells for 72 h.The growth inhibition rate of P.donghaiense was higher than70%at 96 h,and the growth of cell was still inhibited at the 8th day.The inhibitory effect of G.biloba extract on P.donghaiense was fast.After 24 h treatment,the P.donghaiense in the high concentration group（≥3.0 g/L）basically lost their swimming ability.After 48h treatment,the experimental group with the concentration higher than 2.0 g/L had a significant inhibitory effect on the growth of P.donghaiense（P<0.05）.After 96 h of experimental treatment,all the experimental groups had significant inhibitory effect on the growth of P.donghaiense（P<0.05）.The inhibitory rate of 2.0 g/L G.biloba extract on the growth of P.donghaiense was up to 84.61%.The 96 h-EC₅₀ of C.camphora and G.biloba extract on the growth of P.donghaiense were 1.23 g/L and 1.22 g/L,respectively.（2）The volatile constituents of C.camphora leaves were analyzed by gas chromatography-mass spectrometer.The terpenoids,such as linalool,nerolidol,Spathulenol and 1,8-cineole,which may have allelic effects,were identified.The non-volatile components of G.biloba leaf extract were analyzed by liquid chromatography-mass spectrometry.Catechin,rutin and quercetin,which may have allelopathic effects,were isolated and identified.Nerolidol in C.camphora leaf extract can significantly inhibit the growth of P.donghaiense.The 96 h-EC₅₀ effect of nerolidol on P.donghaiense growth was 1.25 mg/L.When treated for 24 h,nerolidol at all concentrations could significantly inhibit P.donghaiense growth.4.0 mg/L nerolidol could kill a large number of cells and make the cell lose their swimming ability,and the growth inhibition rate of P.donghaiense reached 63.96%.At 96 h,the inhibition rates of nerolidol at 1.0,2.0,4.0 and8.0 mg/L to P.donghaiense were 40.34%,68.72%,99.55%and 99.18%,respectively.Nerolidol is a potential active ingredient for algal inhibition.（3）In order to explore the allelopathic inhibition effect of nerolidol and G.biloba leaf on P.donghaiense,The effects of 1.0 mg/L nerolidol（nerolidol group）,1.0 g/L G.biloba（G.biloba group）and 1.0 mg/L nerolidol+1.0 g/L G.biloba（formula group）on the inhibitory rate,swimming ability,photosynthesis and cell membrane integrity of P.donghaiense were studied.The results showed that the inhibition effect of formula group on most indexes of P.donghaiense was significantly higher than that of nerolidol group and G.biloba group alone（P<0.05）.After 96 h of algal inhibition,the inhibition rates of nerolidol,G.biloba and formula groups were 40.16%,51.39%and 74.75%,respectively.The swimming activity of P.donghaiense in formula group was much lower than that of other groups.The actual photochemical quantum efficiency,photosynthesis efficiency and relative maximal electron transport rates of photosynthetic systemⅡ（PSⅡ）of P.donghaiense in formula group were significantly lower than those in other groups（P<0.05）,and the light saturation coefficient of P.donghaiense in formula was lower than that in control group.In addition,after 48 h,the leakage of nucleic acid(A₂₆₀)and protein(A₂₈₀)in formula group was significantly higher than that of G.biloba group（P<0.05）,and the damage degree of cell membrane was more serious.The results showed that nerolidol and G.biloba extract had synergistic effect on the algal inhibition of P.donghaiense.（4）In order to investigate the toxic effect of formula algal suppressor on fishery economic organisms,the adult Meretrix meretrix was used as the biological test organism for ecotoxicology test of G.biloba-nerolidol formula.During the experiment,there were no dead individuals in all groups.During 0-72 h of the experiment,the extension rate of the breathing tube in 1+1 formula group（1.0 mg/L nerolidol+1.0 g/L G.biloba）and 2+2formula group（2.0 mg/L nerolidol+2.0 g/L G.biloba）was stable at 30%,and the breathing tube of M.meretrix could be quickly retracted after contact,and the shell of the other 70%of the M.meretrix was closed.At 96 h,almost all M.meretrix in the 1+1formula group extended their breathing tubes,but the extended tubes were longer than those in the control group,and the extended tubes could quickly retract after being touched.In the 2+2 formula group,few M.meretrix extended their breathing tubes,and the rest remained confined to the shell.After M.meretrix were treated with the formula algal suppressor for 96 h,M.meretrix were put back into the clean artificial seawater without the addition of drugs.All M.meretrix of each group extended their breathing tubes normally within 24 h,and withdrew quickly after being touched.No dead individuals were observed within 48 h.The results showed that the development of algal suppressor with terrestrial plant source materials has great application potential.The results of this study provide a scientific basis for further exploring the inhibition mechanism of plant allelopathic substances on algae and preparation of formula algal suppressor using plants.