【作者】 刘志强；

【导师】 张利平；

【作者基本信息】 河北大学 ， 微生物学， 2010， 硕士

【摘要】 本实验采用根瘤匀浆法,利用无氮BAP培养基、S培养基、JA培养基、ISP4培养基和FTW培养基,分别从采自云南省、吉林省、内蒙古自治区和海南省的12种植物根瘤内分离得到60株固氮放线菌。分离结果表明,成分相对简单的BAP培养基和S培养基分离效果较好,还发现萘啶酮酸和放线菌酮的组合要比同剂量链霉素和制霉菌素的组合、链霉素和放线菌酮的组合以及萘啶酮酸和制霉菌素的组合的抑菌效果好。对所分离得到的60株菌株进行了形态观察,菌株49615等在基内菌丝上可形成大量的单个孢子;菌株49640等基丝丰富,气丝较短,菌株49641等基丝丰富,有分枝,气丝形成短孢子链;菌株49601等14株菌可产生褐色、黄色、棕色等可溶性色素。生理生化试验结果表明,所有供试菌株均能利用葡萄糖和阿拉伯糖作为唯一碳源生长,除菌株49616外,均能利用木糖作为唯一碳源生长。所有菌株都不能在含有10% NaCl的培养基上生长、不能使纤维素水解、利用七叶苷、不能利用乙酸钠。大多数菌株能使硝酸盐还原、淀粉水解。仅菌株49611、49612、49616能利用柠檬酸钠;仅菌株49601能使明胶液化,菌株49606能利用草酸钠。16S rDNA序列的分析结果表明,60株供试菌被划分为4个属,菌株49601等38株菌属于小单孢菌属(Micromonospora);菌株49639等20株菌属于链霉菌属(Streptomyces);菌株49659属于野野村菌属(Nonomuraea);菌株49660属于假诺卡菌属(Pseudonocardia)。回接实验结果表明,供试菌株49616和49625在回接于相应的宿主植物旱冬瓜(Alnus nepalensis)、马桑(Coriaria sinica)和沙枣(Elaeagnus angustifolia)后,宿主植物的长势好于对照组,且小单孢菌属的菌株49625回接马桑后出现结瘤现象。对植物根瘤切片进行电镜观察表明,根瘤细胞内有菌丝、孢囊等结构,对根瘤总DNA进行弗兰克氏菌属、小单胞菌属、链霉菌属、假诺卡菌属特异性序列扩增,所检测的根瘤中都出现了不同数量的特异性扩增条带,说明同种植物的根瘤中存在多种放线菌,具有物种多样性。更多还原

【Abstract】 In this study, we used root-nodule homogenate method and isolated 60 nitrogen-fixing actinomycetes from root nodules of 12 plants, which were collected from Yunnan, Jilin,Inner Mongolia and Hainan of China. The nitrogen-free media of BAP, S, JA, ISP4 and FTW were used for isolation of strains. The results show that the efficiency of BAP medium and S medium with relatively simple components were much better than other media. We also found that the bacteriostatic effect of the combination of nalidixic acid and cycloheximide was better than the same dose combination of streptomycin and nystatin, streptomycin and cycloheximide and nalidixic acid and nystatin.The morphological observation of the isolated strains show that strain 49615 produced a single spore on substrate mycelium; substrate mycelium of strain 49640 were abundant and aeril mycelium were short; strain 49641 had rich substrate mycelium and short spore chains; Strain 49601 and other 14 strains could produce brown, yellow, brown, or other soluble pigments.The results of physiological and biochemical showed that all of the isolated strains could utilized glucose and arabinose as the sole carbon source. In addition to strain 49616,all of the isolated strains could use xylose as the sole carbon source. All of the strains could not grow in the medium with 10% NaCl; All of the strains could not hydrolyze cellulose. All of the strains could use rescin glycosides.Most of strains could reducte nitrate and hydrolyze starch. All of the strains could not utilize sodium acetate, only strain 49611, strain 49612, strain 49616 were able to use sodium citrate,only strain 49601 could liquefact gelatin and only strain 49606 could use sodium oxalate.The 16S rDNA sequence analysis of the isolated 60 strains showed that the 60 strains were located into 4 genus: strain 49601 and other 37 strains belonged to Micromonospora, strain 49639 and others 19 strains belonged to Streptomyces, strain 49659 belonged to Nonomuraea, Strain 49660 belonged to Pseudonocardia.The results of nodulation experiments showed that the host plants Alnus nepalensis, Coriaria sinica and Elaeagnus angustifolia growed better than the control group and the Coriaria sinica grewed nodules after the infection of strain 49616 and strain 49625,which belonged to Micromonospora. The mycelium of actinomycetes and other structures in root nodule were found by observing the ultramicrocut, and also amplified specific sequences in the total DNA of nodules by using specific primers of Frankia , Micromonospora, Streptomyces, and Pseudonocardia , as a result,there were different specific bands were getten , which showed that there were different kinds of actinomycetes strains and species diversity in 12 root nodules.更多还原