川西高原药用植物内生放线菌的分离鉴定及抑菌活性研究
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摘要
放线菌是一类重要的微生物资源,放线菌产生的大量生理活性物质,可作为抗菌剂、酶与酶制剂、抗病毒、抗肿瘤剂的来源。目前人们已完成了大量抗生素筛选工作,放线菌产生的新抗生素越难被发现,因此开始从不同的材料中寻找新的抗生素。植物中的内生放线菌与宿主在进化历程中长期共生,形成了独特的遗传和代谢体系,内生放线菌能够产生结构新颖、生物活性多样的次生代谢产物,药用植物内生放线菌在医药、农林、环境保护及工业等领域具有广阔的应用前景。
本实验对药用植物内生放线菌的分离进行研究。利用4种选择性培养基,9种碳源,对采自四川西部高原地区的10种药用植物进行内生放线菌的分离。共计分离214株内生放线菌。不同植物,不同分离部位获得的内生放线菌数目均有较大差异。各种培养基的分离效果不同。改良高氏二号培养基和腐殖酸培养基分离效果较好。培养基抑制剂组合为：50μl/ml重铬酸钾和100μl/ml放线菌酮。这组抑制剂能够较好的抑制杂菌,对放线菌的生长影响较小。
选择50株内生放线菌,检测其对3种植物病原菌抑菌效果,结果表明大多数内生放线菌能不同程度的抑制植物病原菌的生长。40株内生放线菌对植物病原菌产生抑制,17株内生放线菌对至少两种植物病原菌有抑制作用,其中Y3101和Y3104,对三种植物病原菌均有抑制作用。这两株菌分离自鼠尾草。抑菌试验说明供试药用植物内生放线菌菌株能够拮抗植物病原菌,具有产生新的活性物质的潜力。
通过形态特征、培养特征对内生放线菌类群进行分析,发现大多数内生放线菌为链霉菌属(Streptomyces)。对14株抑菌效果较好的内生放线菌,结合表型特征观察以及16S rDNA全序列分析,进行了初步鉴定。通过构建系统发育树,初步确定菌株的分类地位：Y3056被鉴定为黑色链霉菌(Streptomyces niger), Y3057为浅多色链霉菌(Streptomyces pluricolorescens), Y3066为天蓝色链霉菌(Streptomyces coelicolor), Y3069为桑普逊链霉菌(Streptomyces sampsonii)，Y3078为土味链霉菌(Streptomyces odor ifer), Y3083为娄彻氏链霉菌(Streptomyces rochei), Y3087为细黄链霉菌(Streptomyces microflavus), Y3095为环圈链霉菌(Streptomyces anulatus), Y3099为淤泥链霉菌(Streptomyces limosus), Y3101为褶皱链霉菌(Streptomyces plicatus),Y3103为杀真菌素链霉菌(Streptomycesfungicidicus), Y3104为微白黄链霉菌(Streptomyces albidoflavus), Y3106为沙场链霉菌(Streptomyces arenae), Y3109为脱叶链霉菌(Streptomyces exfoliatus)。
Actinomycete is an important microbiological resource which can produce lots of bio-control agents such as antibacterial agents, enzymes and enzyme preparations, antivirus and antitumor activities. By now, plenty of antibiotics were found, people need to explore a new way to find new antibiotics. As a result of long-held symbiotic relationship between endophytic microbes and plants, endophyte may have unique genetic and biological system. Medical plants represent a rich and relatively unexplored source of endophytic actinomycetes, from the secondary metabolites of endophytic actinomycetes, many compounds with new structures and huge bioactive diversities could be found. Several reports indicated that endophytic actinomycetes seems to have great potential in medical, agricultural, environmental and industrial fields.
In this study, the endophytic actinomycetes were isolated from 10 medical plants collected from Western Sichuan Plateau. Four selective culture media and nine carbon resources were chosen. As a result,214 isolates of endophytic actinomycetes were obtained. Different experimental materials on the endophytic actinomycetes obtained in quantity is quite different. The isolation effect of different media are mixed ragged. The result indicated that modified Gause II medium and HVA medium are best for isolation in this experiment. The proper inhibitor combination: 50μl/ml potassium dichromate and 100μl/ml cycloheximide.
Fifty representative isolates were tested for antifungal activities from three kinds of plant pathogens. The results indicated that most of isolates have antifungal activities.Forty strains inhibited at least one of the plant pathogens tested. Seventeen strains inhibited two plant pathogens. Strain Y3101 and Strain Y3104 inhibited three plant pathogens used in this experiment. They were both isolated from Salvia officinalis. The result suggested that the endophytic actinomycetes of medicinal plant were potential inhibitor for plant pathogen, it can be a new source of novel bioactive compounds.s
According to morphology and culture properties, the result showed that most of the isolates were streptomycete strains. Fourteen strains were chosen for 16S rDNA sequences analysis, then reconstruct phylogeny tree. Based on the result of morphological and molecular identification, taxonomic status of these strains were preliminarily determined.The isolates Y3056,Y3057,Y3066,Y3069,Y3078,Y3087,Y3095,Y3099,Y3101,Y3103,Y3104,Y3106 were identified as Streptomyces niger, Streptomyces pluricolorescens, Streptomyces coelicolor, Streptomyces sampsonii, Streptomyces odorifer, Streptomyces rochei, Streptomyces microflavus, Streptomyces anulatus, Streptomyces limosus, Streptomyces plicatus, Streptomyces fungicidicus, Streptomyces albidoflavus, Streptomyces arenae, Streptomyces exfoliates, respectively.
本实验对药用植物内生放线菌的分离进行研究。利用4种选择性培养基,9种碳源,对采自四川西部高原地区的10种药用植物进行内生放线菌的分离。共计分离214株内生放线菌。不同植物,不同分离部位获得的内生放线菌数目均有较大差异。各种培养基的分离效果不同。改良高氏二号培养基和腐殖酸培养基分离效果较好。培养基抑制剂组合为：50μl/ml重铬酸钾和100μl/ml放线菌酮。这组抑制剂能够较好的抑制杂菌,对放线菌的生长影响较小。
选择50株内生放线菌,检测其对3种植物病原菌抑菌效果,结果表明大多数内生放线菌能不同程度的抑制植物病原菌的生长。40株内生放线菌对植物病原菌产生抑制,17株内生放线菌对至少两种植物病原菌有抑制作用,其中Y3101和Y3104,对三种植物病原菌均有抑制作用。这两株菌分离自鼠尾草。抑菌试验说明供试药用植物内生放线菌菌株能够拮抗植物病原菌,具有产生新的活性物质的潜力。
通过形态特征、培养特征对内生放线菌类群进行分析,发现大多数内生放线菌为链霉菌属(Streptomyces)。对14株抑菌效果较好的内生放线菌,结合表型特征观察以及16S rDNA全序列分析,进行了初步鉴定。通过构建系统发育树,初步确定菌株的分类地位：Y3056被鉴定为黑色链霉菌(Streptomyces niger), Y3057为浅多色链霉菌(Streptomyces pluricolorescens), Y3066为天蓝色链霉菌(Streptomyces coelicolor), Y3069为桑普逊链霉菌(Streptomyces sampsonii)，Y3078为土味链霉菌(Streptomyces odor ifer), Y3083为娄彻氏链霉菌(Streptomyces rochei), Y3087为细黄链霉菌(Streptomyces microflavus), Y3095为环圈链霉菌(Streptomyces anulatus), Y3099为淤泥链霉菌(Streptomyces limosus), Y3101为褶皱链霉菌(Streptomyces plicatus),Y3103为杀真菌素链霉菌(Streptomycesfungicidicus), Y3104为微白黄链霉菌(Streptomyces albidoflavus), Y3106为沙场链霉菌(Streptomyces arenae), Y3109为脱叶链霉菌(Streptomyces exfoliatus)。
Actinomycete is an important microbiological resource which can produce lots of bio-control agents such as antibacterial agents, enzymes and enzyme preparations, antivirus and antitumor activities. By now, plenty of antibiotics were found, people need to explore a new way to find new antibiotics. As a result of long-held symbiotic relationship between endophytic microbes and plants, endophyte may have unique genetic and biological system. Medical plants represent a rich and relatively unexplored source of endophytic actinomycetes, from the secondary metabolites of endophytic actinomycetes, many compounds with new structures and huge bioactive diversities could be found. Several reports indicated that endophytic actinomycetes seems to have great potential in medical, agricultural, environmental and industrial fields.
In this study, the endophytic actinomycetes were isolated from 10 medical plants collected from Western Sichuan Plateau. Four selective culture media and nine carbon resources were chosen. As a result,214 isolates of endophytic actinomycetes were obtained. Different experimental materials on the endophytic actinomycetes obtained in quantity is quite different. The isolation effect of different media are mixed ragged. The result indicated that modified Gause II medium and HVA medium are best for isolation in this experiment. The proper inhibitor combination: 50μl/ml potassium dichromate and 100μl/ml cycloheximide.
Fifty representative isolates were tested for antifungal activities from three kinds of plant pathogens. The results indicated that most of isolates have antifungal activities.Forty strains inhibited at least one of the plant pathogens tested. Seventeen strains inhibited two plant pathogens. Strain Y3101 and Strain Y3104 inhibited three plant pathogens used in this experiment. They were both isolated from Salvia officinalis. The result suggested that the endophytic actinomycetes of medicinal plant were potential inhibitor for plant pathogen, it can be a new source of novel bioactive compounds.s
According to morphology and culture properties, the result showed that most of the isolates were streptomycete strains. Fourteen strains were chosen for 16S rDNA sequences analysis, then reconstruct phylogeny tree. Based on the result of morphological and molecular identification, taxonomic status of these strains were preliminarily determined.The isolates Y3056,Y3057,Y3066,Y3069,Y3078,Y3087,Y3095,Y3099,Y3101,Y3103,Y3104,Y3106 were identified as Streptomyces niger, Streptomyces pluricolorescens, Streptomyces coelicolor, Streptomyces sampsonii, Streptomyces odorifer, Streptomyces rochei, Streptomyces microflavus, Streptomyces anulatus, Streptomyces limosus, Streptomyces plicatus, Streptomyces fungicidicus, Streptomyces albidoflavus, Streptomyces arenae, Streptomyces exfoliates, respectively.
引文
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