Algicidal Bacteria Associated With Harmful Bloom-forming Karenia Mikimotoi In Estuarine Soil And Study On The Algicidal Effect And Mechanism

Harmful algal blooms is able to destroy the marine ecosystem and the water environment.It also pose a serious threat to human health and the national economy.It is an important research direction to find effective methods to prevent and control red tide.Some studies have shown that there are microorganisms in the marine environment that can significantly inhibit the growth of red-tide microalgae and show great potential in the prevention and control of harmful red tide.In this context,this paper screened out the bacteria with inhibitory effect on red-tide microalgae Karenia mikimotoi from seawater and estuary soil,explored the growth inhibitory effect,mode and mechanism.The main research results are as follows:1.Isolation and identification algicidal bacteria association with K.mikimotoiThrough co-culture experiments,9 strains of algicidal bacteria were screened from the marine environment,and 5 strains of algicidal bacteria were screened from the estuary soil.Species information of the strains were determined by 16 S rRNA sequencing,and phylogenetic trees were constructed.Among them,the marine bacteria were Alteromonas sp.,Halomonas sp.,Marinobacter sp.,Paracoccus sp.,Rhodobacteraceae,Idiomarina sp..The soil strains were Kocuria roses,Pseudoalteromonas sp.,and Flavobaterium sp.,the algicidal rate could reach over 90% after 48 hours,both Pseudoalteromonas sp.and Flavobaterium sp.inhibited algae in an indirect way.2.The physiological inhibition mechanisms of the extracellular active substances of Pseudoalteromonas sp.on the growth of K.mikimotoi(1)After co-culturing the sterile filtrate of Pseudoalteromonas sp.with K.mikimotoi,the growth rate of algal cells was better than that of the control group under the condition of 2% filtrate volume.With the increase of filtrate volume,the growth of algal cells was inhibited,4% filtrate volume as the "minimum inhibitory concentration" and 8% filtrate volume as the "saturation effect concentration".The number and morphology of algal cells were observed under an inverted fluorescence microscope,and it was found that algal cells gradually broke down into cell fragments and showed yellow-green color under green fluorescence with the increase of filtrate volume and the extension of action time.(2)The flow cytometry was used to detect the cell cycle of the algae,and it was found that 4% of the filtrate volume acted for 12 h,algal cell cycle was arresed at G2 phase.When the filtrate volume increased to 8%,the genomic DNA fragment of the algae cells was lost,and 30% Sub-G1 peak was displayed.Therefore,it was speculated that under the action of short time and low concentration of filtrate,the algae cell cycle was arrested at G2 phase.Under the action of high concentration for a long time,DNA fragments of algal cells were lost,and might occur algal cell apoptosis.(3)Under the stress of 8% volume filtrate,ROS accumulated in algal cells,and the ROS level was the highest after 3 hours of treatment.At this time,it was observed that the algal cells were completely presented red color with irregular morphological changes by fluorescence microscope.The ROS level decreased after 6 hours of treatment,and the algal cells ruptured and presented yellow-green color.(4)Under different filtrate volume stress,Caspase-3 enzyme activity enhanced and reached the highest level at 6 h.After the addition of activated oxygen inhibitor NAC,it was found that the activity of Caspase-3 enzyme was significantly reduced,indicating that the accumulation of ROS could activate the Caspase-3 enzyme.(5)Flow cytometry showed that the apoptosis of algal cells occurred under the treatment of sterile filtrate,and the apoptosis rate increased with the increase of filtrate.In addition,the apoptosis rate increased with the prolongation of treatment time.It was speculated that the activation of Caspase-3 enzyme may lead to the occurrence of apoptosis,and the apoptosis rate of algal cells was reduced after the addition of activated oxygen inhibitor NAC and Caspase-3 enzyme inhibitor Ac-DEVD-CHO,indicating that the ROS mediated Caspase-3 enzyme-dependent apoptosis pathway was involved in the apoptosis of algal cells caused by bacterial filtrate.