Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (11): 225-236.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0288
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LUO Ya-jun1(
), SUN Hong-min2, HE Ning2, YUAN Li-jie1(), XIE Yun-ying2()
Received:2021-03-11
Online:2021-11-26
Published:2021-12-03
Contact:
YUAN Li-jie,XIE Yun-ying
E-mail:592062203@qq.com;yuanlijie1970@163.com;xieyy@imb.pumc.edu.cn
LUO Ya-jun, SUN Hong-min, HE Ning, YUAN Li-jie, XIE Yun-ying. Isolation and Antibacterial Activity of Actinomycetes from the Nodules and Rhizosphere Soil of Hippophae rhamnoides in Tibet[J]. Biotechnology Bulletin, 2021, 37(11): 225-236.
| 采样地点 Sampling location | 样品种类 Sample type | 编号 No. | 海拔 Altitude/m | 采样时间 Sampling time | 坐标 Coordinates |
|---|---|---|---|---|---|
| 林芝南伊沟区 | 沙棘根瘤 | P1 | 2 999 | 2019. 09. 21 | 29o08’25.15” N,94o12’58.71” E |
| 根际土壤 | E1 | 2 999 | 2019. 09. 21 | 29o08’25.15” N,94o12’58.71” E | |
| 山南市错那县 | 大沙棘根瘤 | P2 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E |
| 根际土壤 | E2 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E | |
| 山南市错那县 | 小沙棘根瘤 | P3 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E |
| 根际土壤 | E3 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E | |
| 山南市错那县 | 沙棘根瘤 | P4 | 4 255 | 2019. 09. 23 | 28o15’18.76” N,91o45’47.52” E |
| 根际土壤 | E4 | 4 255 | 2019. 09. 23 | 28o15’18.76” N,91o45’47.52” E | |
| 山南市隆子县 | 沙棘根瘤 | P5 | 3 915 | 2019. 09. 23 | 28o25’12.01” N,92o23’48.99” E |
| 根际土壤 | E5 | 3 915 | 2019. 09. 23 | 28o25’12.01” N,92o23’48.99” E |
Table 1 Sample collection information
| 采样地点 Sampling location | 样品种类 Sample type | 编号 No. | 海拔 Altitude/m | 采样时间 Sampling time | 坐标 Coordinates |
|---|---|---|---|---|---|
| 林芝南伊沟区 | 沙棘根瘤 | P1 | 2 999 | 2019. 09. 21 | 29o08’25.15” N,94o12’58.71” E |
| 根际土壤 | E1 | 2 999 | 2019. 09. 21 | 29o08’25.15” N,94o12’58.71” E | |
| 山南市错那县 | 大沙棘根瘤 | P2 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E |
| 根际土壤 | E2 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E | |
| 山南市错那县 | 小沙棘根瘤 | P3 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E |
| 根际土壤 | E3 | 4 515 | 2019. 09. 23 | 28o18’48.24” N,91o49’06.59” E | |
| 山南市错那县 | 沙棘根瘤 | P4 | 4 255 | 2019. 09. 23 | 28o15’18.76” N,91o45’47.52” E |
| 根际土壤 | E4 | 4 255 | 2019. 09. 23 | 28o15’18.76” N,91o45’47.52” E | |
| 山南市隆子县 | 沙棘根瘤 | P5 | 3 915 | 2019. 09. 23 | 28o25’12.01” N,92o23’48.99” E |
| 根际土壤 | E5 | 3 915 | 2019. 09. 23 | 28o25’12.01” N,92o23’48.99” E |
| 培养基Culture medium | 培养基成分Medium composition |
|---|---|
| PDB | 成品,购于北京奥博星生物技术公司,pH 7.2 |
| ISP 2 | 酵母膏4 g,麦芽糖10 g,葡萄糖4 g,pH 7.2 |
| FM3 | 可溶性淀粉20 g,甘油5 g,脱脂小麦胚芽10 g,肉提取物3 g,干酵母3 g,CaCO3 3 g,pH 7.0 |
| FM4 | 半乳糖3.3 g,水化糊精3.3 g,甘油1.7 g,豆胨1.7 g,玉米浆0.83 g,CaCO3 2 g,（NH4）2SO4 0.33 g,pH 7.0 |
| FM5 | 葡萄糖10 g,牛肉膏10 g,蛋白胨1 g,NaCl 5 g,pH 7.0 |
| M331 | 葡萄糖20 g,普通淀粉5 g,蛋白胨 10 g,（NH4）2SO4 7 g,CaCO3 2 g,pH自然 |
| F1 | 普通淀粉20 g,葡萄糖20 g,蛋白胨3 g,牛肉膏3 g,微量元素1 mL,CaCO3 2 g,花生饼粉10 g,pH 7.2 |
| M2 | 甘露醇40 g,麦芽浸粉40 g,酵母浸膏粉10 g,K2HPO4 2 g,MgSO4·7H2O 0.5 g,FeSO4·7H2O 0.01 g,pH 7.2 |
| Cazpeck | K2HPO4 1 g,NaNO3 0.3 g,KCl 0.005 g,MgSO4·7H2O 0.005 g,FeSO4 0.001 g,蔗糖30 g,pH 7.0 |
| FM10 | 甘油20 g,糖蜜10 g,酪蛋白5 g,蛋白胨1 g,CaCO3 4 g,pH 7.2 |
| FM11 | 葡萄糖20 g,麦芽浸粉40 g,酵母浸粉4 g,K2HPO4 5 g,NaCl 2.5 g,CaCO3 0.4 g,ZnSO4 0.04 g,pH 6.0 |
| YMS | 酵母浸粉4 g,麦芽浸粉10 g,可溶性淀粉4 g,pH 自然 |
Table 2 12 kinds of fermentation medium
| 培养基Culture medium | 培养基成分Medium composition |
|---|---|
| PDB | 成品,购于北京奥博星生物技术公司,pH 7.2 |
| ISP 2 | 酵母膏4 g,麦芽糖10 g,葡萄糖4 g,pH 7.2 |
| FM3 | 可溶性淀粉20 g,甘油5 g,脱脂小麦胚芽10 g,肉提取物3 g,干酵母3 g,CaCO3 3 g,pH 7.0 |
| FM4 | 半乳糖3.3 g,水化糊精3.3 g,甘油1.7 g,豆胨1.7 g,玉米浆0.83 g,CaCO3 2 g,（NH4）2SO4 0.33 g,pH 7.0 |
| FM5 | 葡萄糖10 g,牛肉膏10 g,蛋白胨1 g,NaCl 5 g,pH 7.0 |
| M331 | 葡萄糖20 g,普通淀粉5 g,蛋白胨 10 g,（NH4）2SO4 7 g,CaCO3 2 g,pH自然 |
| F1 | 普通淀粉20 g,葡萄糖20 g,蛋白胨3 g,牛肉膏3 g,微量元素1 mL,CaCO3 2 g,花生饼粉10 g,pH 7.2 |
| M2 | 甘露醇40 g,麦芽浸粉40 g,酵母浸膏粉10 g,K2HPO4 2 g,MgSO4·7H2O 0.5 g,FeSO4·7H2O 0.01 g,pH 7.2 |
| Cazpeck | K2HPO4 1 g,NaNO3 0.3 g,KCl 0.005 g,MgSO4·7H2O 0.005 g,FeSO4 0.001 g,蔗糖30 g,pH 7.0 |
| FM10 | 甘油20 g,糖蜜10 g,酪蛋白5 g,蛋白胨1 g,CaCO3 4 g,pH 7.2 |
| FM11 | 葡萄糖20 g,麦芽浸粉40 g,酵母浸粉4 g,K2HPO4 5 g,NaCl 2.5 g,CaCO3 0.4 g,ZnSO4 0.04 g,pH 6.0 |
| YMS | 酵母浸粉4 g,麦芽浸粉10 g,可溶性淀粉4 g,pH 自然 |
Fig. 1 Phylogenetic tree of strain N-J based on gene sequences of representative strains of 16S rRNA genera The value at the bifurcation point is obtained by 1 000 times of self-xpanding analysis. Scale 0.020 evolution distance
Fig. 2 Diversity comparison of actinomycetes isolated from 7 media
Fig. 3 Diversity distribution of actinomycetes in seabuck-thorn root nodules and rhizosphere soil samples
Fig. 4 Antibacterial activities of fermentation products of 46 representative strains A:Staphylococcus aureus（ATCC 29213）. B:Methicillin resistant S. aureus（MRSA）181223. C:Pseudomonas aeruginosa（ATCC 27853）. D:Escherichia coli（ATCC 25922）. E:Klebsiella pneumoniae（ATCC 700603）. F:Carbapenem resistant Acinetobacter baumannii（crab）181236. G:Candida albicans（ATCC 10231）. Heatmap:In the 96 well plates of 7 kinds of bacteria,the bacteria liquid fermented in 12 kinds of media was added to each well,and the growth time was 24 h. In the figure,green indicates good antibacterial activity,red indicates almost no antibacterial activity,and the transition from red to green to light purple green indicates that the fermentation broth has little or no antibacterial activity against the tested bacteria. The medium of 1-12 is PDB,ISP 2,FM3,FM4,FM5,M331,F1,M2,Cazpeck,FM10,FM11,and YMS
| 发酵菌株 Fermentation strain | 菌株发酵液对检定菌最大抑制率及发酵培养基 The maximum inhibition rate of fermentation broth to the tested bacteria and fermentation medium | ||||||
|---|---|---|---|---|---|---|---|
| 金黄色葡萄球菌ATCC 29213 | 耐甲氧西林金黄色葡萄球菌MRSA 181223 | 铜绿假单胞菌 ATCC 27853 | 大肠埃希菌ATCC 25922 | 肺炎克雷伯菌ATCC 700603 | 耐碳青霉烯鲍曼不动杆菌 CRAB 181236 | 白色念珠菌ATCC 10231 | |
| Streptomyces sp. XZ19-363 | 87.4%（FM4） | 82.7%（FM4） | 87.1%（FM4） | < 50% | 50.8%（FM4） | 91.3%（FM4） | < 50% |
| Streptomyces sp. XZ19-323 | 86.9%（FM3） | 79.6%（FM3） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-034 | 84.6%（Cazpeck） | 73.1%（Cazpeck） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-175 | 86.2%（FM3） | 79.6%（FM3/FM4） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-147 | 86.4%（FM4） | 81.0%（FM4） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-130 | 88.8% （ISP 2） | 83.4%（ISP 2） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-336 | 88.1%（ISP 2） | 83.1%（ISP 2） | < 50% | < 50% | < 50% | < 50% | 78.9%（FM3） |
| Streptomyces sp. XZ19-098 | 88.3%（FM3） | 82.7% （ISP 2/FM4） | < 50% | 72.6%（YMS） | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-152 | 84.1%（PDB） | 82.7%（FM4） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-091 | 88.1%（FM3） | 83.1%（FM4） | < 50% | < 50% | < 50% | < 50% | 83.8%（FM3） |
| Streptomyces sp. XZ19-122 | 88.3%（FM3） | 83.4%（ISP 2） | < 50% | < 50% | < 50% | 53.0%（F1） | 84.1%（FM3） |
| Streptomyces sp. XZ19-015 | 88.3% （ISP 2） | 83.7%（ISP 2） | < 50% | < 50% | < 50% | 64.4%（FM3） | 83.8%（FM4） |
| Streptomyces sp. XZ19-030 | 88.3% （ISP 2） | 82.0%（FM4） | < 50% | < 50% | < 50% | < 0.5% | 81.0%（M2） |
| Streptomyces sp. XZ19-036 | 87.4% （ISP 2） | 81.7%（ISP 2） | < 50% | < 50% | < 50% | < 0.5% | 85.0% （FM11） |
| Streptomyces sp. XZ19-093 | < 50% | < 50% | < 50% | < 50% | < 50% | 55.1%（M331） | < 50% |
| Streptomyces sp. XZ19-021 | < 50% | < 50% | < 50% | < 50% | < 50% | 55.7%（F1） | < 50% |
| Streptomyces sp. XZ19-006 | 77.3%（M2） | 71.3%（M2） | < 50% | < 50% | < 50% | 51.2%（M331） | < 50% |
| Streptomyces sp. XZ19-003 | 82.2%（YMS） | 65.5%（YMS） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Promicromonospora sp. XZ19-011 | < 50% | < 50% | < 50% | < 50% | < 50% | 73.9%（M331） | < 50% |
| Micromonospora sp. XZ19-205 | 59.4%（YMS） | < 50% | < 50% | < 50% | < 50% | < 50% | < 50% |
| 美罗培南 | 87.1% | 9.50% | 91. 5% | 91.6% | 93.5% | 42.2% | - |
| 万古霉素 | 83.3% | 74.1% | -1.2% | 37.8% | 15.1% | 40.0% | - |
| 氟康唑 | - | - | - | - | - | - | 66.1% |
| 伊曲康唑 | - | - | - | - | - | - | 44.5% |
| 两性霉素B | - | - | - | - | - | - | 64.6% |
Table 3 Inhibition rate of fermentation extracts of some strains to the tested strains
| 发酵菌株 Fermentation strain | 菌株发酵液对检定菌最大抑制率及发酵培养基 The maximum inhibition rate of fermentation broth to the tested bacteria and fermentation medium | ||||||
|---|---|---|---|---|---|---|---|
| 金黄色葡萄球菌ATCC 29213 | 耐甲氧西林金黄色葡萄球菌MRSA 181223 | 铜绿假单胞菌 ATCC 27853 | 大肠埃希菌ATCC 25922 | 肺炎克雷伯菌ATCC 700603 | 耐碳青霉烯鲍曼不动杆菌 CRAB 181236 | 白色念珠菌ATCC 10231 | |
| Streptomyces sp. XZ19-363 | 87.4%（FM4） | 82.7%（FM4） | 87.1%（FM4） | < 50% | 50.8%（FM4） | 91.3%（FM4） | < 50% |
| Streptomyces sp. XZ19-323 | 86.9%（FM3） | 79.6%（FM3） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-034 | 84.6%（Cazpeck） | 73.1%（Cazpeck） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-175 | 86.2%（FM3） | 79.6%（FM3/FM4） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-147 | 86.4%（FM4） | 81.0%（FM4） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-130 | 88.8% （ISP 2） | 83.4%（ISP 2） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-336 | 88.1%（ISP 2） | 83.1%（ISP 2） | < 50% | < 50% | < 50% | < 50% | 78.9%（FM3） |
| Streptomyces sp. XZ19-098 | 88.3%（FM3） | 82.7% （ISP 2/FM4） | < 50% | 72.6%（YMS） | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-152 | 84.1%（PDB） | 82.7%（FM4） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Streptomyces sp. XZ19-091 | 88.1%（FM3） | 83.1%（FM4） | < 50% | < 50% | < 50% | < 50% | 83.8%（FM3） |
| Streptomyces sp. XZ19-122 | 88.3%（FM3） | 83.4%（ISP 2） | < 50% | < 50% | < 50% | 53.0%（F1） | 84.1%（FM3） |
| Streptomyces sp. XZ19-015 | 88.3% （ISP 2） | 83.7%（ISP 2） | < 50% | < 50% | < 50% | 64.4%（FM3） | 83.8%（FM4） |
| Streptomyces sp. XZ19-030 | 88.3% （ISP 2） | 82.0%（FM4） | < 50% | < 50% | < 50% | < 0.5% | 81.0%（M2） |
| Streptomyces sp. XZ19-036 | 87.4% （ISP 2） | 81.7%（ISP 2） | < 50% | < 50% | < 50% | < 0.5% | 85.0% （FM11） |
| Streptomyces sp. XZ19-093 | < 50% | < 50% | < 50% | < 50% | < 50% | 55.1%（M331） | < 50% |
| Streptomyces sp. XZ19-021 | < 50% | < 50% | < 50% | < 50% | < 50% | 55.7%（F1） | < 50% |
| Streptomyces sp. XZ19-006 | 77.3%（M2） | 71.3%（M2） | < 50% | < 50% | < 50% | 51.2%（M331） | < 50% |
| Streptomyces sp. XZ19-003 | 82.2%（YMS） | 65.5%（YMS） | < 50% | < 50% | < 50% | < 50% | < 50% |
| Promicromonospora sp. XZ19-011 | < 50% | < 50% | < 50% | < 50% | < 50% | 73.9%（M331） | < 50% |
| Micromonospora sp. XZ19-205 | 59.4%（YMS） | < 50% | < 50% | < 50% | < 50% | < 50% | < 50% |
| 美罗培南 | 87.1% | 9.50% | 91. 5% | 91.6% | 93.5% | 42.2% | - |
| 万古霉素 | 83.3% | 74.1% | -1.2% | 37.8% | 15.1% | 40.0% | - |
| 氟康唑 | - | - | - | - | - | - | 66.1% |
| 伊曲康唑 | - | - | - | - | - | - | 44.5% |
| 两性霉素B | - | - | - | - | - | - | 64.6% |
Fig. 5 Comparison of fermentation activities of 12 kinds of fermentation media
Fig. 6 Analysis of metabolites of 3 actinomycetes
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