Evaluation On The Activity Of Sponge-epiphytic Actinomyces And Their Secondary Metabolites Against Plant Pathogens

Marine actinomycetes have unique properties in terms of physiological traits and genetic background due to the special environment,thus they have great potential to produce specific metabolites.In this study,Streptomyces malaysiens was isolated from Siphonochalina truncata in the Xisha Islands for the first time and then the antimicrobial activity of Streptomyces malaysiens was studied.Finally,the secondary metabolites of the strain were isolated,purified and identified,in order to provide a new efficient and green method for agricultural disease control.The main contents are as follows:1.Study on the antimicrobial activity of biocontrol actinomycetesAn active actinomycete strain was isolated and purified from sponge samples collected from Xisha Islands,Hainan,and named ITBB-ZK-a5;with 16S r RNA gene sequencing,the strain was identified as Streptomyces malaysiens by combining the constructed phylogenetic tree and colony morphology.The results of antibacterial activity showed that the strain had good fungistatic effect against 19strains of pathogenic fungi such as Colletotrichum musae and Fusarium oxysporum f.sp.cubense（FOC）.And it also showed some fungistatic effect on 7 pathogenic bacteria.The strain ITBB-ZK-a5 had good generational stability against Colletotrichum musae.2.Study on antimicrobial activity of fermentation extract and fermentation broth of strain ITBB-ZK-a5The optimal fermentation medium for strain ITBB-ZK-a5 was rice fermentation medium,and the extracts with 15-20 d of fermentation showed the best antimicrobial activity;Its fermented extracts showed better photo-stability and thermal stability.And its EC₅₀for Colletotrichum musae and Colletotrichum gloeosporioides were38.88μg/m L and 11.70μg/m L respectively.The biological control of FOC was tested on strain ITBB-ZK-a5 fermentation broth under laboratory potted conditions.Strain ITBB-ZK-a5 had obvious control effect on FOC,but its control effect and control mechanism in field need to be confirmed.3.Study on isolation and activity of secondary metabolites of strain ITBB-ZK-a5The whole genome sequencing of strain ITBB-ZK-a5 showed that the genome of the strain consisted of 1 contig with a size of 11,465,825 bp and a G+C content of71.07%.Analyzing the biosynthesis gene cluster types of the secondary metabolites of the strain ITBB-ZK-a5 genome by using anti SMASH,there were 50 potential biosynthesis gene clusters of the secondary metabolites with the main types being NPRS,PKS and terpene.The fermented extracts of the strain ITBB-ZK-a5 were separated to obtain 5pure compounds with the use of normal-phase silica gel,reversed-phase silica gel,Sephadex LH20 gel chromatographic column,semi-preparative liquid chromatographic separation,nuclear magnetic resonance and other spectroscopic techniques.As a result,5 compounds were identified as ansamycin compounds,including 4 new compounds and 1 known compound,compound 1 was reblastatin with new configuration,compound 2 was KOS 1806 with new configuration,compound 3 was a known compound herbimycin H,compound 4 was 7-carbamate of geldanamycin,and compound 5 was a new ansamycin-type compound.The activity screening of these 5 compounds showed that compounds 1 and 2 had weak inhibitory effects on Micrococcus luteus,compounds 3 and 4 had inhibitory effects on Vibrio alginolyticus and other tested pathogenic bacteria,while the MIC values of compounds 3 and 4 against Micrococcus luteus were lower than that of kanamycin.