부산대학교 도서관

海洋放线菌因其所处环境特殊,在生理性状和遗传背景方面均具有独特的性质,因此海洋放线菌具有产生特殊代谢产物的巨大潜力.本研究以西沙海绵为研究对象,挖掘其具有抑菌活性的共附生放线菌资源.首先,利用平板分离法从海绵样品中分离纯化出一株活性放线菌菌株ITBB-ZK-a5,对其进行 16S rRNA基因测序,结合构建的系统发育树和菌落形态特征,确定该菌株为链霉菌.其次,采用平板对峙法研究菌株ITBB-ZK-a5 对植物病原真菌的抑菌广谱性和持效性.结果表明:该菌株对供试的 16 株病原真菌均有较好的抑菌活性,抑菌谱较广,且对峙 14、30 d仍具有活性,抑菌持效性较好;为了进一步验证该菌株平板通过产生活性物质抑制病原菌生长,对菌落边缘的无菌琼脂块进行抑菌活性研究,结果表明抑菌率与菌落距离呈负相关,说明放线菌可能通过向平板中分泌活性物质以抑制病原菌的生长.该菌株连续转接 5 代后,仍具有较好的世代稳定性.再次,利用大米固体培养基对菌株ITBB-ZK-a5 进行发酵,乙酸乙酯萃取浓缩得到提取物,通过滤纸片法对发酵粗提物进行光稳定性和热稳定性研究,结果表明,随着水浴温度升高和紫外照射时间的增加,菌株ITBB-ZK-a5 对香蕉炭疽病病原菌的抑制效果呈下降趋势,但仍具有较强的抑菌活性.最后,测定菌株 ITBB-ZK-a5 对香蕉炭疽病病原菌的半最大效应浓度(EC50)为 38.88 μg/mL,对木瓜炭疽病病原菌的EC50 值为 11.70 μg/mL.本研究对南海海绵共附生放线菌进行了分离鉴定,筛选出具有抑制多种热带作物病原真菌活性的生防菌株ITBB-ZK-a5,并对其进行抑菌活性研究,为后续生防菌株的开发利用奠定基础.
Marine actinomycetes possess great capacity in producing special metabolites due to their special environ-ment and unique properties in physiological characters and genetic background.In this study,sponge was used as isola-tion material to explore the resources of symbiotic actinomycetes with antibacterial activity.Firstly,an active actino-mycetes strain,ITBB-ZK-a5,was isolated and purified from the sample of Xisha sponge by plate separation method.The strain was identified as Streptomyces by analysis of the phylogenetic tree based on 16S rRNA gene sequence and colony morphological characteristics.Secondly,the inhibitory spectrum of strain ITBB-ZK-a5 against plant pathogenic fungi was studied by plate confrontation method.The results showed that ITBB-ZK-a5 had a broad antibacterial spec-trum,in which the 16 tested pathogenic fungi were inhibited significantly.ITBB-ZK-a5 also showed good antibacterial activity in 14 days and 30 days.In order to explore the reason why strain ITBB-ZK-a5 can inhibit the growth of patho-genic bacteria,the antibacterial activity of asepsis agar from the ITBB-ZK-a5 edge were determined,and the results showed that the antibacterial rate were negatively correlated with colony distance.This result revealed that actinomy-cetes maybe can secrete active substances to inhibit the growth of pathogenic bacteria.Thirdly,the strain had a good antibacterial stability by detecting the antimicrobial activity of five continuously dynasties.Again,light stability and thermal stability of the ITBB-ZK-a5 crude extracts from the rice solid fermentation were tested through the filter method;the results show that with the increase of bath temperature and UV duration,the antibacterial activity of the strain ITBB-ZK-a5 crude extracts were decreased.Finally,the EC50 value of strain ITBB-ZK-a5 against anthrax diseases of banana and papaya were determined.Plant diseases infected by fungi are the main agricultural diseases,and chemical pesticides are the most commonly used method in diseases control.However,the widespread use of chemical pesticides will not only enhance crop resistance to drugs,but also cause pollution to the ecological environment.More importantly,pesticide residues also have a certain influence on human health.Therefore,the common goal of the world is to find more green,efficient and safe control methods.Actinomycetes are found to have biological control effects.The symbi-otic actinomycetes isolated from sponges have an inhibitory effect on pathogenic fungi of tropical crops,which can be applied to efficient diseases control,conforming to the concept of green environmental protection.In this paper,we isolated and identified the strain ITBB-ZK-a5,symbiotic actinomycetes from South China Sea sponge,which has the activity of inhibiting pathogenic fungi of various tropical crops,and studied its antibacterial activity,providing a re-search basis for the development and utilization of biocontrol strains in the future.