北仑河红树林内生放线菌分离、鉴定及活性研究

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摘要放线菌是天然抗生素的重要来源，是寻找天然抗感染活性产物的出发菌。为了分离广西壮族自治区北仑河红树植物内生放线菌，检测其抗菌活性及初步确定活性菌株的分类地位，本研究采集5种红树植物共15份样品，用14种液体培养基富集培养4周后，涂布于相应固体培养基进行分离纯化；以6种细菌和20株白色念珠菌(Candida albicans)为指示菌，采用点植法和滤纸片法对分离到的菌株进行抗菌活性检测；选取其中有抗菌活性的菌株进行16S rRNA基因测序，在属水平初步确定其分类地位。结果表明，共分离到菌株200株，其中从白骨壤(Avicennia marina)、秋茄(Kandelia candel)、红海榄(Rhizophora stylosa)、苦郎树(Clerodendrum inerme)和海漆(Excoecaria agallocha)中分别获得62、43、38、29和28株。根据培养和形态学特征初步剔除重复后得到106株，其中活性菌株52株，阳性率为49.1%，包括21株链霉菌属(Streptomyces)、26株拟诺卡氏菌属(Nocardiopsis)、2株假诺卡氏菌属(Pseudonocardia)、2株壤球菌属(Agrococcus)和1株栖白蚁菌属(Isoptericola)。其中菌株UMBR 1016和UMBR 1034与已知菌株Nocardiopsis alba DSM 43377T的相似度分别为98.27%和98.37%；菌株UMBR 1008与Nocardiopsis flavescens SA6T的相似度为98.36%，菌株UMBR 1028与Streptomyces pratensis ch24T的相似度为98.49%，这4株内生放线菌的相似度都低于98.6%，可能为拟诺卡氏菌属和链霉菌属的潜在新种。有38株对至少一株细菌有抑菌作用，35株对至少一株白色念珠菌有抑菌作用，阳性率分别达到35.8%和33.0%。广西北仑河红树植物内生放线菌资源丰富，抗菌活性较高，值得深入研究。研究结果将促进我国红树林植物资源的保护与红树植物内生放线菌药用资源的深入开发。

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	杨桂柳
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关键词 ：红树植物, 内生放线菌, 分离, 抗菌活性

Abstract： Actinomycetes, as important sources of naturally occurred antibiotics, are strains for discovery of natural anti-infective agents. In order to isolate and purify the endophytic actinomycetes from mangrove plants in Beilun Estuary National Nature Reserve, Guangxi Zhuang Autonomous Region, explore their potential antimicrobial activities against pathogenic bacteria and fungi, and preliminarily clarify taxonomic status of bioactive strains, all 15 samples from different parts of 5 mangrove plants were cultured separately in 14 different liquid media for 4 weeks，and the enriched cultural media were then spreaded on the corresponding agar media with standard serial dilution plating method to isolate and purify the colonies. A total of 200 isolates were purified from samples including 62 strains from Avicennia marina, 43 strains from Kandelia candel，38 strains from Rhizophora stylosa, 29 strains from Clerodendrum inerme and 28 strains from Excoecaria agallocha, respectively. After removing the duplicate isolates by comparison of their cultural and morphological characterizations, 106 purified endophytic strains were tested for their antimicrobial activities against 6 strains of bacteria and 20 strains of Candida albicans by point planted method and disk diffusion method. A total of 52 isolates showed positive results and the positive rate was 49.1%. Among them, inhibitory rates against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Proteusbacillus vulgaris, Hemolytic streptococcus and Klebsiella pneumoniae were 16%, 15.1%, 4.7%, 7.5%, 11.3% and 3.8%, respectively; Inhibitory rates aganist drug-sensitive strains of Candida albicans and drug-resistant strains of Candida albicans were 16.0% and 29.2%, respectively. The bioactive strains were identified preliminarily as Streptomyces (21 strains), Nocardiopsis (26 strains), Pseudonocardia (2 strains), Agrococcus (2 strains) and Isoptericola (1 strain) by phylogenetic analysis based on 16S rRNA gene sequences. Among them, the similarities of both strains UMBR1016 and UMBR1034 with Nocardiopsis alba DSM 43377T were 98.27% and 98.37%, respectively; the similarity between strain UMBR 1008 and Nocardiopsis flavescens SA6T, and between strain UMBR 1028 and Streptomyces pratensis ch24T, was 98.36% and 98.49%, respectively. The results that similarities of 16S rRNA gene sequences were less than 98.6% indicated 3 strains including UMBR1016, UMBR1034 and UMBR 1008 were potential new species of Nocardiopsis genus and UMBR 1028 was potential new species of Streptomyces genus. The 38 and 35 isolates were found to show antimicrobial activity against at least one tested bacteria and one strain of Candida albicans, and the inhibitory rates were 35.8% and 33.0%, respectively. Abundant endophytic actinomycetes, including new species and potential antimicrobial activities found in the study, indicated that endophytic actinomycetes from mangrove plants in Beilun of Guangxi province could be a potential source for the discovery of new species and natural products and deserved further development in next step. The study will improve the conservation of mangrove plants in our country and further utilization of endophytic actinomycetes as important sources of drug discovery.

Key words： Mangrove plants Endophytic Actinomycetes Isolation Antibacterial activity

收稿日期: 2014-12-18 出版日期: 2015-05-13

ZTFLH:

Q939

基金资助:国家自然科学基金;广西自然科学基金;广西红树林保护重点实验室基金

通讯作者: 姜明国 E-mail: mzxyjiang@163.com

引用本文:

杨桂柳杨立芳姜明国吴家法甘光华庹利孙承航. 北仑河红树林内生放线菌分离、鉴定及活性研究[J]. , 2015, 23(7): 894-904.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I7/894

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