| Karenia mikimotoi that belongs to Karenia,Dinophyceae is one of the toxic red tide algal species.K.mikimotoi can secrete ichthyotoxic and hemolytic toxins,which threaten or kill marine organisms.There exist notable limitations such as inefficiency and secondary pollution in traditional physical and chemical methods,eco-friendly and efficient biological controls based on algicidal bacteria show high application potentials,and are getting worldwide attentions.In this paper,we investigated the microbial community inhabiting K.mikimotoi phycosphere and the succession during algal growth,the responses of K.mikimotoi and the phycosphere community to nutrients stimulation and exogenous algicidal strain Pseudoalteromonas sp.QF1.QF1 were isolated from estuarine of Yellow river,China,and can effectively inhabit the growth of K.mikimotoi by producing algicidal metabolites.The main contents and results are as follows: 1.Identification of QF1 and its influence on the growth and morphology of sterile K.mikimotoi(1)Identification,morphological observation and cultivation of QF1In this study,we identified QF1 based on 16 S rRNA gene sequence,observed bacterial morphology with dilution separation methods and SEM,monitored the growth curve of QF1 in 2216 E medium and fresh water medium containing 1%(v/v)or 3%(v/v)NaCl.The results showed that QF1 belonged to Pseudoalteromonas sp.,colonies were white round,opaque,neat edge and smooth in surface,cells were rod-shaped of about 1.0 ?m long by 0.2 ?m wide.The strain could grow rapidly in fresh water medium and 2216 E medium,which entered logarithmic phase and stable growth phase after culture for 2 hours and 20 hours,respectively.(2)The influence of QF1 on the growth and morphology of sterile K.mikimotoiAfter co-culturing QF1 fermentation broth with sterile K.mikimotoi,high concentrations of QF1 fermentation broth caused mass mortality of K.mikimotoi.After 3 days co-cultivation,the algicidal rate of 3%(v/v)QF1 fermentation broth treated group was more than 90%.3% QF1 fermentation broth obviously impacted on cell movement and made algae agglomerate together.Under the stress of QF1,remarkable changes for algae cells could be observed.Obvious wrinkle occurred in algae cells,and cell membrane of K.mikimotoi broke down,algae cell content was leakaged,a number of bacteria attached to the surface of algae.The TEM results indicated that plasmolysis began to appear in K.mikimotoi cells after 1 day co-cultivation and some chloroplasts concentrated to the middle of the algae cell with volume increase.2.K.mikimotoi associated with phycosphere microbial community and its responses to nutrients stimulation and algicidal strain Pseudoalteromonas sp.QF1(1)Composition of the microbial community inhabiting K.mikimotoi phycosphereA large diversity of microorganisms inhabited in K.mikimotoi phycosphere.The microorganisms inhabiting K.mikimotoi phycosphere were mainly affiliated with Alphaproteobacteria and Sphingobacteriia.At the genus level,an unidentified genus that belongs to Rhodobacteraceae and Phaeodactylibacter that belongs to Saprospiraceae were dominant,which accounted for 32.9-57.8% and 10.7-30.0% of the microbial communities.In addition,Pseudoalteromonas was detected in K.mikimotoi phycosphere,which only accounted for 0-2.7%.The succession of the community in phycosphere was also observed during the cultivation process: the relative abundances of the unidentified Rhodobacteraceae genus and Halomonas decreased,while Phaeodactylibacter,Maritimibacter,Pseudoalteromonas and Alteromonas increased.(2)Responses of the phycosphere microbial community to nutrients additionAfter nutrients stimulation,a high mortality of K.mikimotoi was observed.Microbial communities analysis indicated that Alphaproteobacteria(11.36-62.71%)and Sphingobacteriia(0.83-17.29%)significantly decreased,while Gammaproteobacteria(4.36-85.44%)markedly increased.At the genus level,the relative abundance of the genus that belongs to Rhodobacteraceae(6.86-39.22%)and Phaeodactylibacter(0.83-17.23%)decreased,while Halomonas(1.54-55.13%)and Alteromonas(0.23-41.57%)became dominated populations.In addition,Pseudoalteromonas also slightly increased after nutrients stimulation.There were different successions of microbial communities in 3% and 6% nutrients stimulated groups.In 3% nutrients stimulated group,Halomonas and Alteromonas decreased,while Phaeodactylibacter and Maritimibacter obviously increased at the 9th day.In 6% nutrients stimulated group,Halomonas and Alteromonas always dominated during the cultivation process.(3)Responses of the phycosphere microbial community to QF1Pseudoalteromonas predominated in both 3% and 6% QF1 fermentation broth treated groups at the early phase of co-cultivation.In 3% QF1 fermentation broth treated group,the relative abundance of Pseudoalteromonas decreased to 1.10-9.50% at the 9th day,while the genus that belongs to Rhodobacteraceae,Phaeodactylibacter and Maritimibacter increased to 37.10-39.49% ? 28.86-29.45% and 6.19-6.80%,respectively.The microbial communities at the 9th day in 3% nutrients stimulated groups showed a higher similarity with those in the control group.Differ with 3% QF1 treated group,the unidentified Rhodobacteraceae genus and Phaeodactylibacter in 6% QF1 treated group did not obviously increase at the 9th day,even Pseudoalteromonas significantly decreased like 3% QF1 treated group.Summarily,this study indicated that diverse microbial populations inhabited in K.mikimotoi phycosphere and had a dynamic change during the cultivation process.Nutrients stimulation could stimulate the growth of Halomonas and Alteromonas and result in a high mortality of K.mikimotoi.QF1 fermentation broth caused massive death of K.mikimotoi and substantial shifts in structure of the microbial community.Furthermore,the microbial community in K.mikimotoi phycosphere showed robustness to low-dose nutrients and QF1 disturbances.The results could benefit our understanding about the relationships of microalgae and microorganism,prefect the theory construction and provide the theory basis for practical application of controlling harmful red tides. |
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