Isolation And Characterization Of A Multifunctional Algicidal Streptomyces Sp.Strain Against Microcystis Aeruginosa

With the intensification of water eutrophication and global warming,harmful cyanobacteria blooms(HCB)have increasingly become a global concern.Harmful cyanobacterial blooms can destroy aquatic ecosystems by degrading water quality,consuming oxygen from water bodies,and producing harmful cyanotoxins,which threaten the survival of aquatic organisms and human health.Microcystis is one of the most dominant genus of cyanobacterial blooms in freshwater bodies,and also the main producer of cyanotoxin microcystins(MCs).The control of HCB with microbes(algicidal bacteria)has become a research focus because of its low cost,simple operation,environmental friendliness.But there are still many probems remained to be solved before algicidal bacteria could be applied in field scale.In this paper,to improve the field application possibility of algicidal bacteria,a definition of multifunctional algicidal bacteria was firstly proposed,i.e.bacteria have not only algicidal activity but also biological activity against other harmful microbes and MCs-degrading or inhibition activity,and not limited to these activities.A Streptomyces sp.strain with high algicidal activity against Microcystis aeruginosa was screened from an anti-Fusarium graminearum actinomyces libriary.This strain was designated as HG-16.HG-16.HG-16 also inhibited the synthesis of MCs strongly.The algicidal characteristics,basic characteristics of algicidal substance(s),algicidal mechanisms and genomic analysis of HG-16 were investigated.The main results are listed below.1.Isolation and identification of multi-functional algicidal actinomycetes.In this part,actinomycete strains with antagonistic activity against F.graminearum was isolated by plate confrontation method from the purple soil sampled in Chongqing,and the algicidal activity of these actinomycetes was subsequently determined by co-culture with M.aeruginosa.A strain,designated as HG-16 showed the highest algicidal activity.The inhibition zones during plate confrontation culture showed HG-16 also suppressed the growth of F.solanis effectively.The colonies of strain HG-16 grew on the Gause's synthetic agar medium were circular and dry.The aerial mycelia were white,and pinkish grey spores were produced after prolonged incubation.The spore chains were rectiflexibilis and the spore was short rod-shaped with smooth surface.Combined with morphological and 16 S rDNA gene sequence analysis,HG-16 was identified as S.amritsarensis.2.Studies on the algicidal characteristics of S.amritsarensis HG-16: HG-16 showed high algicidal activity against different Microcystis species and strains,such as M.aeruginosa NIES-843,NIES-90 and NIES-44,and several other common harmful cyanobacteria such as Phormidium sp.FACHB-1099,Anabaena flos-aquae sp.FACHB-1255,and Oscillatoria sp.FACHB-868;The algicidal acitivity of HG-16 was not affected by the associated bacteria of cyanobacteria;HG-16 secreted algicidal substance(s)during the stationary growth phase,and attacked the algae in an indirect way by these active compound(s);The strain HG-16 showed high algicidal activity against to M.aeruginosa FACHB-905 of different cell densities.The algicidal activity of HG-16 was light dependent and the photosynthesis system was one of the targets of HG-16,and the damages caused by S.amritsarensis HG-16 could not be repaired during the dark culture period.S.amritsarensis HG-16 secreted algicidal substance has acid and alkali resistance,but has high temperature sensitivity;The algicidal activity of cell-free filtrate was not affected by proteinase K treatment,suggesting that the major algicidal substance(s)secreted by HG-16 were not protein(s).3.Studied on the algicidal mechanisms of HG-16 against M.aeruginosa:the cell morphological changes of M.aeruginosa after being treated with the cell-free filtrate and cultures of HG-16,suggesting that the M.aeruginosa cells were bound by actinomycete hyphae and killed by active compounds released by HG16;the cell-free filtrate of HG-16 treatment resulted in the quick decrease of Fv/Fm of M.aeruginosa cells,implying that the photosynthetic capacity of those cells was inhibited;Compared with the control,the ROS content in M.aeruginosa cells increased at first and then decreased,indicating that HG-16 caused oxidative stress of cyanovacterial cells;The results of RT-Qpcr(Real-time quantitative PCR)further indicates that S.amritsarensis HG-16 seriously affected the expression of photosynthesis system related genes and strongly inhibited the expression of microcystins synthesis key gene.The whole genome of S.amritsarensis HG-16 was sequenced by GridION sequencing platform;7 presumble plasmids exist in HG-16.The genomic DNA sequence was analyzed with antiSMASH on line,and 24 gene clusters,including 11 NRPS/PKS(non-ribosomal peptide synthetases/polyketide synthases);These gene clusters are responsible for the synthesis of lanthipeptide,terpene,siderophore peptides and polyketides.A T2 PKS synthesis gene cluster is on a 21 kb plasmid,and it might synthesize 4 kinds of new Type ? polyketides;These gene clusters might encode some new algicidal substances secreted by HG-16,but further verification is needed.