大连海域沉积物中可培养海洋链霉菌活性及其多样性

Activity and diversity of culturable Streptomyces isolated from marine sediments of sea areas of Dalian

以大肠杆菌、金黄色葡萄球菌和尖孢镰刀枯萎病菌作为测试靶目标,采用9种分离培养基从大连海域13个不同采样点的海洋沉积物样品中分离到165株海洋链霉菌。从165株海洋放线菌中筛选到对金黄色葡萄球菌具有抑制活性的菌株85株,占总菌株数的51.5%;对大肠杆菌具有抑制活性的菌株27株,占总菌株数的16.4%;对尖孢镰刀枯萎病菌具有抑制活性的菌株仅有6株,占总菌株数的3.6%。因此,海洋链霉菌的活性更多地表现为对细菌的抗性,尤其对革兰氏阳性细菌具有更高的抑制活性。对其中具有抑制活性或形态独特的菌株进行了16S r DNA序列分析,并构建系统发育树,显示活性海洋链霉菌具有丰富的种类多样性和广谱抗菌活性。同种海洋链霉菌与土壤链霉菌活性比较结果也表明,海洋链霉菌多表现抗革兰氏阳性细菌活性。

Actinomycetesare an important source of microorganisms used in medicine. Tens of thousands of biologically active substances of microbial origin have been found,about 45% of which are produced by actinomycetes. The marine environment has high salinity,high pressure,low temperature,low nutritive content and other characteristics,so that the population characteristics and metabolic pathways of marine actinomycetes are different from those of terrestrial actinomycetes,and can be a source of novel species and metabolites. In total,165 antimicrobial isolates were screened from13 marine sediment samples collected in the seas around Dalian using nine different media. The results showed that improved humic acid vitamin agar medium（ HV-2） and glycerol arginine agar medium（ M2） were the best types of medium for isolating marine actinomycetes,followed by improved LSE-SE-2 agar medium（ LSE-SE-2） and oat agar medium（ OM）.Using agar block methods,the antagonistic activity of the 165 strains against three pathogens（ Fusariumoxysporium f sp.cucumarinum,Staphylococcus aureus and Escherichia coli） was analyzed in vitro. Among the 165 isolates,95 strains showed antagonistic activity against these three test pathogens. Of the 165 isolates,85（ 51.5%） strains showed activity against S.aureus. The diameter of the inhibition zone of four of the 85 strains was more than 30 mm; 27 strains（ 16.4% of the total isolates） showed activity against E. coli,and the diameter of the inhibition zone of strains YH6 and YH21 against E. coli was more than 27 mm. Six strains（ M95,M108,HV14,HL9,HE16,HE66）,3. 6% of the total isolates,showed activity against F. oxysporumf sp. cucumarinum. The results indicated that marine actinomycetes often show antagonistic activity against bacteria,especially Gram-positive bacteria.Meanwhile,16 S r DNA sequences of interesting strains with either specialphenotypes or strong antimicrobial activity were analyzed. Phylogenetic analysis using 16 S r DNA sequencing revealed that marine Streptomyces showed abundant species diversity and broad activity. Apart from the hygroscopicus group,95 marine Streptomyces showed antagonistic activity covering all groups of Streptomyces. The 95 marine Streptomyces belong to different groups such as the roseosporusgroup, golden group, albosporus group, cinereus group, yellow group, blue group,lavendulae group,black group,griseorubroviolaceus group,griseofuscus group and green group. According to the Sci Finder substances library,the biological activity of recorded species from the NCBI library,which had sequences approximately homologous to the 95 marine Streptomyces isolates,was analyzed. The 95 marine Streptomyces showed bioactivity,but 23 strains among the recorded species failed to exhibit activity against the pathogens. Meanwhile,13 isolates from the 95 marine Streptomyces exhibited bioactivity,whereas the 13 corresponding recorded species failed to exhibit activity. This suggested that different habitats give the same species different activities. The same species isolated from marine sediments and soil showed different activities. Streptomyces species from marine sediments often showed antagonistic activity against Gram-positive bacteria,and had low antagonistic activity against fungi and Gram-negative bacteria. Through the analysis of the antagonistic activity of marine Streptomyces,the results will help researchers to improve our understanding of microbial diversity,and the isolates obtained will also help us to search for metabolites with novel activity.