| In recent years, the rapidly industrialization of the coastal cities has influencedthe marine environment severely. The frequent harmful algal blooms (HABs), notonly post a threat to the sustainable development of the marine economy, but alsoaffect the physical and mental health of human. In order to prevent and control theharmful algal blooms many countermeasures have been proposed. Algicidal bacteriawere found capable of inhibiting or degrading HABs. The potential of security andhigh efficiency of algicidal bacteria provide new ideas and methods for controllingthe HABs, which will be widely applied to control the red tides in future.In this study, two bacterial strains of BS02and BS03, were isolated from theZhangjiang Estuary Mangrove National Natural Reserve, China. They exhibitingstrong activity against the toxic dinoflagellate, Alexandrium tamatense. Then thestudy of lysing characteristics of the algicidal bacteria against A. tamatense wascarried out. In addition, the lysing mechanism of strain BS02was discussed from thestructure of algal cells, antioxidant system, photosynthetic system and the level ofgene expression. Finally, column chromatography, mass spectrometry and nuclearmagnetic resonance techniques were used to identify the main algicidal compounds,secreted by algicidal bacterium BS02. These findings indicated that algicidalbacterium could be a potential bio-agent applied in controlling HABs in the future.The main conclusions obtained from the study are shown below:(1) Two algicidal bacteria strains were successfully isolated, Based on16SrRNA gene sequences, biochemical and morphological characteristics, the strainBS02and BS03were identified to be vibrio and Microbulbifer.(2) The algicidal effect of two strains exhibit concentration-dependent, theremoved rate of the A.tamarense have been up to85%treated with2.0%(v/v)concentration for4days, while algal growth was prompted at0.25%and0.5%bacterial concentrations. Moreover, the lysing experiment showed that both BS02andBS03strains were attacked A. tamatense indirectly by extracellular algicidal substances rather than the bacteria themselves.(3) There are certain species-specific of the strains BS02and BS03, sincethem displayed varying degrees of lysing activities against the eighteen algae tested.Results showed that the strain BS02could greatly inhibit the growth of the algae, theChlorella (CH), Skeletonema tropicum (ST), Phaeodactylum (PT), Chlorellaautotrophica (CA), Asterionella japonica (AJ), Dicrateria inornata (DI), Platymonassubcordlformls (PS), Alexandrium minutum (AMTW) and Scrippsiella trochoidea(STXM01), there was no significant lytic effect was observed in the rest of testedalgae. Besides, the strain BS03exhibits strong algicidal effects to the algae,Chaetoceros compressus (CC), Chattonella marina (CM), Prorocentrum.donghaiense(PD), Thalassiosira weissflogii (TW), Skeletonema costatum (SC), Platymonashelgolandica (PH), Amphiprora alata (AA) and Alexandrium catenella (ACDH),without any algicidal effect to other tested algae species.(4) The A. tamatense treated with algicidal bacterium BS02supernatants, werecaused significant changes of the structure, such as plasmolysis, the fragmentation ofthe cell wall, membrane dissolution, and deformation of the structure of other majorcell organelles，such as chloroplasts, mitochondria and nuclei. Associated with thesechanges in cell structure, a variety of physiological and biochemical indicators alsochanged in algal cells. Because of an increase of reactive oxygen species (ROS) inalgal cells, the activities of the antioxidant enzymes superoxide dismutase (SOD),catalase (CAT) were enhanced under ROS stress. Levels of malodialdehyde (MDA)were significantly higher than those of the controls. The decrease of the content ofphotosynthetic pigments of algal cells and the values of the photosyntheticfluorescence efficiency Fv/Fm showed directly that the photosynthesis of the algalcells have been obviously damaged. Caspase-3protease activities of algal cellsincreased when treated with strain BS02supernatants, which suggested the algae mayexperience a process of programmed cell death (PCD).(5) The transcription levels of the psbA, psbD, cob and cox genes were detectedby Real-Time PCR. The psbA and psbD genes have been reported activelyparticipating in the synthesis of the D1and D2protein in the reaction center of PSII, and the other two genes cob and cox were involved in the cell pigment metabolism.The Results showed that the transcriptional level of the psbA, cob and cox genes weredown-regulated, in contrast the psbD gene was up-regulated. In the whole, thephenomenon detected indicated that PSII reaction center was damaged, accompaniedby blocking electron transport, suppressing synthesis of the D1protein denovo,ultimately disrupting the growth of algal cells.(6) The algicidal compounds produced by bacterium BS02could benon-proteinaceous, non-nucleate, non-polysaccharide. The molecular weight of thecompounds were less than1kDa, heat tolerant, stable in acidic and alkalineconditions. And the MALDI-TOF-MS analysis showed that the responsible algicidalcompound was a molecular weight of254(254m/z), its structure was elucidated by13C-and1H-NMR techniques analysis, all these results indicated this mainlycompound corresponded with palmitoleic acid, a white oil, which is a kind ofmonounsaturated fatty acid with16carbon atoms, the molecular formula is C16H30O2.Algicidal activity assay showed that the palmitoleic acid with a moderate activityagainst A.tamarense, and a significant growth-inhibiting effects on A. tamarense atconcentration of20μg/mL. The inhibitory effects increased, even caused algal cellsdeath with the increasing concentration of the palmitoleic acid. |
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