海绵共附生菌Streptomyces olivaceus LHW2444的分离鉴定及其抗青枯菌活性代谢产物研究  

Marine sponge symbiotic Streptomyces olivaceus LHW2444:isolation,identification,and active metabolites against Ralstonia solanacearum

作　　者：孙煜炜 蒋林 李蕾 伍斗生 蒋赛[4] 罗祥超 林厚文[2] 朱伟明[1] SUN Yuwei;JIANG Lin;LI Lei;WU Dousheng;JIANG Sai;LUO Xiangchao;LIN Houwen;ZHU Weiming(School of Medicine and Pharmacy,Ocean University of China,Qingdao 266003,China;Department of Pharmacy,Research Center for Marine Drugs,State Key Laboratory of Oncogenes and Related Genes,Ren Ji Hospital,School of Medicine,Shanghai Jiaotong University,Shanghai 200127,China;College of Biology,Hunan Key Laboratory of Plant Functional Genomics and Developmental Regulation,Hunan University,Changsha 410082,China;School of Pharmacy,TCM and Ethnomedicine Innovation&Development International Laboratory,Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]中国海洋大学医药学院,山东青岛266003 [2]上海交通大学医学院附属仁济医院药学部,癌基因与相关基因国家重点实验室海洋药物研究中心,上海200127 [3]湖南大学生物学院,植物功能基因组学与发育调控湖南省重点实验室,湖南长沙410082 [4]湖南中医药大学药学院,中医药民族医药国际联合实验室,湖南长沙410208

出　　处：《中国海洋药物》2023年第4期1-7,共7页Chinese Journal of Marine Drugs

基　　金：国家自然科学基金项目(82104055,22137006);中国博士后科学基金项目(2020M671163);湖南省自然科学基金项目(2021JJ40058)资助。

摘　　要：目的从海绵中分离培养放线菌,筛选具有抗青枯菌活性的菌株,分离鉴定活性代谢产物。方法以来源于南海西沙永兴岛附近海域的海绵Leucetta chagosensis为实验材料,采用3种选择性分离培养基分离海绵的共附生放线菌,利用16S rRNA序列分析对各菌株进行种属鉴定。对各菌株的发酵提取物进行抗青枯菌活性筛选,采用硅胶柱层析、Sephadex LH-20凝胶柱层析和高效液相色谱法对筛选到的活性菌株的发酵产物进行分离、纯化,运用核磁共振(NMR)、质谱(MS)等手段,鉴定化合物的结构。采用微量稀释法测定化合物的最低抑菌浓度(MIC)。结果分离培养海绵共附生放线菌16株,筛选得到1株具有较好抗青枯菌活性的菌株Streptomyces olivaceus LHW2444,并从该菌株的发酵产物中分离鉴定2个吡咯类化合物、1个苯并二恶茂类化合物、2个吡喃酮类化合物,分别为吡咯-2-甲酰胺(1)、吡咯-2-甲酸(2)、1,3-苯并二噁茂-2-酮-4-甲酰胺(3)、杀菌素B(4)、杀菌素C(5),化合物1~5为首次从该种属放线菌分离得到。首次发现pyrrole-2-carboxylic acid对青枯菌具有较强抑制作用,MIC值为8μg/mL。结论海绵共附生菌Streptomyces olivaceus LHW2444是潜在的植物青枯病生防菌,pyrrole-2-carboxylic acid是抗青枯菌的活性代谢产物。Objective To isolate and culture actinomycetes from marine sponges,screen for strains with activities against Ralstonia solanacearum,and identify the active metabolites.Methods The marine sponge Leucetta chagosensis from adjacent waters of the Yongxing Island of Xisha,South China Sea was used as experimental material.Actinomycetes isolated by using three selective isolation culture medium were identified by 16S rRNA sequence analysis.The fermentation broth of each strain was screened for activities against R.solanacearum.Silica gel column chromatography,Sephadex LH-20 gel column chromatography and high performance liquid chromatography were used to isolate and purify compounds from the fermented products of the active strain.The structures of the compounds were identified by means of nuclear magnetic resonance(NMR)and mass spectrometry(MS).The minimal inhibitory concentration(MIC)of the compounds were determined by microdilution method.Results 16 actinomycetes strains were isolated and cultured,and Streptomyces olivaceus LHW2444 with activity against R.solanacearum was obtained.Two pyrroles,one benzodioxole and two pyrones were isolated and identified from fermentation products of the strain LHW2444,and their structures were elucidated as pyrrole-2-carboxamide(1),pyrrole-2-carboxylic acid(2),1,3-benzodioxole-2-one-4-carboxylamide(3),germicidin B(4)and germicidin C(5).Compounds 1-5 were isolated from Streptomyces olivaceus for the first time.Pyrrole-2-carboxylic acid was found to show strong inhibitory effect on R.solanacearum for the first time,with a MIC value of 8μg/mL.Conclusion Marine sponge symbiotic Streptomyces olivaceus LHW2444 is a potential biocontrol actinomycetes against plant bacterial wilt,and pyrrole-2-carboxylic acid is an active metabolite against R.solanacearum.

关 键 词：青枯菌 海绵 放线菌 代谢产物 抗菌活性 

分 类 号：R915[医药卫生—微生物与生化药学]